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Autosomal dominant polycystic kidney disease is caused by mutations in the membrane receptor PKD1 or the cation channel PKD2. TACAN (also termed TMEM120A), recently reported as an ion channel in neurons for mechanosensing and pain sensing, is also distributed in diverse non-neuronal tissues, such as kidney, heart and intestine, suggesting its involvement in other functions. In this study, we found that TACAN is in a complex with PKD2 in native renal cell lines. Using the two-electrode voltage clamp in Xenopus oocytes, we found that TACAN inhibits the channel activity of PKD2 gain-of-function mutant F604P. TACAN fragments containing the first and last transmembrane domains interacted with the PKD2 C- and N-terminal fragments, respectively. The TACAN N-terminus acted as a blocking peptide, and TACAN inhibited the function of PKD2 by the binding of PKD2 with TACAN. By patch clamping in mammalian cells, we found that TACAN inhibits both the single-channel conductance and the open probability of PKD2 and mutant F604P. PKD2 co-expressed with TACAN, but not PKD2 alone, exhibited pressure sensitivity. Furthermore, we found that TACAN aggravates PKD2-dependent tail curvature and pronephric cysts in larval zebrafish. In summary, this study revealed that TACAN acts as a PKD2 inhibitor and mediates mechanosensitivity of the PKD2-TACAN channel complex. KEY POINTS: TACAN inhibits the function of PKD2 in vitro and in vivo. TACAN N-terminal S1-containing fragment T160X interacts with the PKD2 C-terminal fragment N580-L700, and its C-terminal S6-containing fragment L296-D343 interacts with the PKD2 N-terminal A594X. TACAN inhibits the function of the PKD2 channel by physical interaction. The complex of PKD2 with TACAN, but not PKD2 alone, confers mechanosensitivity.

This retrospective on the voltage-sensing mechanisms and gating models of ion channels begins in 1952 with the charged gating particles postulated by Hodgkin and Huxley, viewed as charges moving across the membrane and controlling its permeability to Na

Regulation of intracellular pH (pH(i)) in cardiomyocytes is crucial for cardiac function; however, currently known mechanisms for direct or indirect extrusion of acid from cardiomyocytes seem insufficient for energetically-efficient extrusion of the massive H(+) loads generated under in vivo conditions. In cardiomyocytes, voltage-sensitive H(+) channel activity mediated by the HVCN1 proton channel would be a highly efficient means of disposing of H(+), while avoiding Na(+)-loading, as occurs during direct acid extrusion via Na(+)/H(+) exchange or indirect acid extrusion via Na(+)-HCO(3)(−) cotransport. PCR and immunoblotting demonstrated expression of HVCN1 mRNA and protein in canine heart. Patch clamp analysis of canine ventricular myocytes revealed a voltage-gated H(+) current that was highly H(+)-selective. The current was blocked by external Zn(2+) and the HVCN1 blocker 5-chloro-2-guanidinobenzimidazole (ClGBI). Both the gating and Zn(2+) blockade of the current were strongly influenced by the pH gradient across the membrane. All characteristics of the observed current were consistent with the known hallmarks of HVCN1-mediated H(+) current. Inhibition of HVCN1 and the NHE1 Na(+)/H(+) exchanger, singly and in combination, showed that either mechanism is largely sufficient to maintain pH(i) in beating cardiomyocytes, but that inhibition of both activities causes rapid acidification. These results show that HVCN1 is expressed in canine ventricular myocytes and provides a major H(+)-extrusion activity, with a capacity similar to that of NHE1. In the beating heart in vivo, this activity would allow Na(+)-independent extrusion of H(+) during each action potential and, when functionally coupled with anion transport mechanisms, could facilitate transport-mediated CO(2) disposal.

Synapses are enriched in the cytoskeletal protein actin, which determines the shape of the pre- and post-synaptic compartments, organizes the neurotransmitter release machinery, and provides a framework for trafficking of components. In the post-synaptic compartment, interactions with actin or its associated proteins are also critical for the localization and activity of synaptic neurotransmitter receptors and ion channels. Actin binding proteins, including spectrin and alpha-actinin, serve as molecular linkages between the actin cytoskeleton and a diverse collection of receptors, including the NMDA receptor (NMDAR) and voltage gated sodium channels. The actin cytoskeleton can regulate neurotransmitter receptors and ion channels by controlling their trafficking and localization at the synapse and by directly gating receptor channel opening. We highlight evidence that synaptic actin couples physically and functionally to NMDAR and supports its activity. The molecular mechanisms by which actin regulates NMDARs are only just emerging, and recent advancements in light and electron microscopy-based imaging techniques should aide in elucidating these mechanisms.

As in mammals, Drosophila circadian clock neurons display rhythms of activity with higher action potential firing rates and more positive resting membrane potentials during the day. This rhythmic excitability has been widely observed but, critically, its regulation remains unresolved. We have characterized and modelled the changes underlying these electrical activity rhythms in the lateral ventral clock neurons (LNvs). We show that currents mediated by the voltage-gated potassium channels Shaw (Kv3) and Shal (Kv4) oscillate in a circadian manner. Disruption of these channels, by expression of dominant negative (DN) subunits, leads to changes in circadian locomotor activity and shortens lifespan. LNv whole-cell recordings then show that changes in Shaw and Shal currents drive changes in action potential firing rate and that these rhythms are abolished when the circadian molecular clock is stopped. A whole-cell biophysical model using Hodgkin-Huxley equations can recapitulate these changes in electrical activity. Based on this model and by using dynamic clamp to manipulate clock neurons directly, we can rescue the pharmacological block of Shaw and Shal, restore the firing rhythm, and thus demonstrate the critical importance of Shaw and Shal. Together, these findings point to a key role for Shaw and Shal in controlling circadian firing of clock neurons and show that changes in clock neuron currents can account for this. Moreover, with dynamic clamp we can switch the LNvs between morning-like and evening-like states of electrical activity. We conclude that changes in Shaw and Shal underlie the daily oscillation in LNv firing rate.

Key points Mouse chromaffin cells in acute adrenal slices exhibit two distinct spiking patterns, a repetitive mode and a bursting mode.A sodium background conductance operates at rest as demonstrated by the membrane hyperpolarization evoked by a low Na+‐containing extracellular saline.This sodium background current is insensitive to TTX, is not blocked by Cs+ ions and displays a linear I‐V relationship at potentials close to chromaffin cell resting potential. Its properties are reminiscent of those of the sodium leak channel NALCN.In the adrenal gland, Nalcn mRNA is selectively expressed in chromaffin cells.The study fosters our understanding of how the spiking pattern of chromaffin cells is regulated and adds a sodium background conductance to the list of players involved in the stimulus‐secretion coupling of the adrenomedullary tissue. Abstract Chromaffin cells (CCs) are the master neuroendocrine units for the secretory function of the adrenal medulla and a finely‐tuned regulation of their electrical activity is required for appropriate catecholamine secretion in response to the organismal demand. Here, we aim at deciphering how the spiking pattern of mouse CCs is regulated by the ion conductances operating near the resting membrane potential (RMP). At RMP, mouse CCs display a composite firing pattern, alternating between active periods composed of action potentials spiking with a regular or a bursting mode, and silent periods. RMP is sensitive to changes in extracellular sodium concentration, and a low Na+‐containing saline hyperpolarizes the membrane, regardless of the discharge pattern. This RMP drive reflects the contribution of a depolarizing conductance, which is (i) not blocked by tetrodotoxin or caesium, (ii) displays a linear I‐V relationship between −110 and −40 mV, and (iii) is carried by cations with a conductance sequence g Na > g K > g Cs. These biophysical attributes, together with the expression of the sodium‐leak channel Nalcn transcript in CCs, state credible the contribution of NALCN. This inaugural report opens new research routes in the field of CC stimulus‐secretion coupling, and extends the inventory of tissues in which NALCN is expressed to neuroendocrine glands., Key points Mouse chromaffin cells in acute adrenal slices exhibit two distinct spiking patterns, a repetitive mode and a bursting mode.A sodium background conductance operates at rest as demonstrated by the membrane hyperpolarization evoked by a low Na+‐containing extracellular saline.This sodium background current is insensitive to TTX, is not blocked by Cs+ ions and displays a linear I‐V relationship at potentials close to chromaffin cell resting potential. Its properties are reminiscent of those of the sodium leak channel NALCN.In the adrenal gland, Nalcn mRNA is selectively expressed in chromaffin cells.The study fosters our understanding of how the spiking pattern of chromaffin cells is regulated and adds a sodium background conductance to the list of players involved in the stimulus‐secretion coupling of the adrenomedullary tissue.

Stromal interaction molecule (STIM) proteins regulate store‐operated Ca2+ entry (SOCE) in innate and adaptive immune cells and participate in the Ca2+ signals that control the functions of neutrophils, the first line of host defence against bacterial and fungal infections. Loss‐of‐function experiments in animal and cellular models indicate that both STIM1 and STIM2 regulate neutrophil functions, but the complexity of the SOCE machinery and the versatility of neutrophils complicate the evaluation of the results. This review aims to summarize the latest progress in the field, with special attention to the details of the experimental designs. Future study design should aim to improve the standardization of experimental procedures and to provide a more holistic understanding of the role of STIM proteins in neutrophils function.

Throughout postnatal maturation of the mouse inner ear, cochlear hair cells display at least two types of mechanically gated ion channel: normal mechanotransducer (MT) channels at the tips of the stereocilia, activated by tension in interciliary tip links, and anomalous mechanosensitive (MS) channels on the top surface of the cells. The anomalous MS channels are responsible for the reverse‐polarity current that appears in mutants in which normal transduction is lost. They are also seen in wild‐type hair cells around birth, appearing 2 days earlier than normal MT channels, and being down‐regulated with the emergence of the normal channels. We review the evidence that the normal and anomalous channels are distinct channel types, which includes differences in localization, susceptibility to pharmacological agents, single‐channel conductance and Ca2+ permeability. The dichotomy is reinforced by the observation that the anomalous current is absent in cochlear cells of Piezo2‐null mice, even though the normal MT current persists. The anomalous current is suppressed by high intracellular Ca2+, suggesting that influx of the divalent ion via more Ca2+‐permeable normal MT channels inhibits the anomalous channels, thus explaining the temporal relationship between the two. Piezo2‐null mice have largely normal hearing, exhibiting up to 20 dB elevation in threshold in the acoustic brainstem response, so raising questions about the significance of PIEZO2 in the cochlea. Since the anomalous current declines with postnatal age, PIEZO2 may contribute to hair cell development, but it does not underlie the normal MT current. Its role in the development of hearing is not understood.

Trabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humour drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humour outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. This study helps elucidate basic mechanotransduction properties that may contribute to intraocular pressure regulation in the vertebrate eye.Chronic elevations in intraocular pressure (IOP) can cause blindness by compromising the function of trabecular meshwork (TM) cells in the anterior eye, but how these cells sense and transduce pressure stimuli is poorly understood. Here, we demonstrate functional expression of two mechanically activated channels in human TM cells. Pressure-induced cell stretch evoked a rapid increase in transmembrane current that was inhibited by antagonists of the mechanogated channel Piezo1, Ruthenium Red and GsMTx4, and attenuated in Piezo1-deficient cells. The majority of TM cells exhibited a delayed stretch-activated current that was mediated independently of Piezo1 by TRPV4 (transient receptor potential cation channel, subfamily V, member 4) channels. Piezo1 functions as the principal TM transducer of physiological levels of shear stress, with both shear and the Piezo1 agonist Yoda1 increasing the number of focal cell-matrix contacts. Analysis of TM-dependent fluid drainage from the anterior eye showed significant inhibition by GsMTx4. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force-sensing contexts. Piezo1-dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure-dependent outflow suggests potential for a novel therapeutic target in treating glaucoma.

Key points Hair cell mechanoelectrical transducer channels are opened by deflections of the hair bundle about a resting position set by incompletely understood adaptation mechanisms.We used three characteristics to define adaptation in hair cell mutants of transmembrane channel‐like proteins, TMC1 and TMC2, which are considered to be channel constituents.The results obtained demonstrate that the three characteristics are not equivalent, and raise doubts about simple models in which intracellular Ca2+ regulates adaptation.Adaptation is faster and more effective in TMC1‐containing than in TMC2‐containing transducer channels. This result ties adaptation to the channel complex, and suggests that TMC1 is a better isoform for use in cochlear hair cells.We describe a TMC1 point mutation, D569N, that reduces the resting open probability and Ca2+ permeability of the transducer channels, comprising properties that may contribute to the deafness phenotype. Abstract Recordings of mechanoelectrical transducer (MET) currents in cochlear hair cells were made in mice with mutations of transmembrane channel‐like (TMC) protein to examine the effects on fast transducer adaptation. Adaptation was faster and more complete in Tmc2–/– than in Tmc1–/–, although this disparity was not explained by differences in Ca2+ permeability or Ca2+ influx between the two isoforms, with TMC2 having the larger permeability. We made a mouse mutation, Tmc1 p.D569N, homologous to a human DFNA36 deafness mutation, which also had MET channels with lower Ca2+‐permeability but showed better fast adaptation than wild‐type Tmc1+/+ channels. Consistent with the more effective adaptation in Tmc1 p.D569N, the resting probability of MET channel opening was smaller. The three TMC variants studied have comparable single‐channel conductances, although the lack of correlation between channel Ca2+ permeability and adaptation opposes the hypothesis that adaptation is controlled simply by Ca2+ influx through the channels. During the first postnatal week of mouse development, the MET currents amplitude grew, and transducer adaptation became faster and more effective. We attribute changes in adaptation partly to a developmental switch from TMC2‐ to TMC1‐ containing channels and partly to an increase in channel expression. More complete and faster adaptation, coupled with larger MET currents, may account for the sole use of TMC1 in the adult cochlear hair cells., Key points Hair cell mechanoelectrical transducer channels are opened by deflections of the hair bundle about a resting position set by incompletely understood adaptation mechanisms.We used three characteristics to define adaptation in hair cell mutants of transmembrane channel‐like proteins, TMC1 and TMC2, which are considered to be channel constituents.The results obtained demonstrate that the three characteristics are not equivalent, and raise doubts about simple models in which intracellular Ca2+ regulates adaptation.Adaptation is faster and more effective in TMC1‐containing than in TMC2‐containing transducer channels. This result ties adaptation to the channel complex, and suggests that TMC1 is a better isoform for use in cochlear hair cells.We describe a TMC1 point mutation, D569N, that reduces the resting open probability and Ca2+ permeability of the transducer channels, comprising properties that may contribute to the deafness phenotype.

Key points Kv2 currents maintain and regulate motoneuron (MN) repetitive firing properties. Kv2.1 channel clustering properties are dynamic and respond to both high and low activity conditions. The enzyme calcineurin regulates Kv2.1 ion channel declustering. In patholophysiological conditions of high activity, Kv2.1 channels homeostatically reduce MN repetitive firing. Modulation of Kv2.1 channel kinetics and clustering allows these channels to act in a variable way across a spectrum of MN activity states. Abstract Kv2.1 channels are widely expressed in the central nervous system, including in spinal motoneurons (MNs) where they aggregate as distinct membrane clusters associated with highly regulated signalling ensembles at specific postsynaptic sites. Multiple roles for Kv2 channels have been proposed but the physiological role of Kv2.1 ion channels in mammalian spinal MNs is unknown. To determine the contribution of Kv2.1 channels to rat α-motoneuron activity, the Kv2 inhibitor stromatoxin was used to block Kv2 currents in whole-cell current clamp electrophysiological recordings in rat lumbar MNs. The results indicate that Kv2 currents permit shorter interspike intervals and higher repetitive firing rates, possibly by relieving Na+ channel inactivation, and thus contribute to maintenance of repetitive firing properties. We also demonstrate that Kv2.1 clustering properties in motoneurons are dynamic and respond to both high and low activity conditions. Furthermore, we show that the enzyme calcineurin regulates Kv2.1 ion channel clustering status. Finally, in a high activity state, Kv2.1 channels homeostatically reduce motoneuron repetitive firing. These results suggest that the activity-dependent regulation of Kv2.1 channel kinetics allows these channels to modulate repetitive firing properties across a spectrum of motoneuron activity states.

Key points There are two subtypes of trimeric intracellular cation (TRIC) channels but their distinct single‐channel properties and physiological regulation have not been characterized. We examined the differences in function between native skeletal muscle sarcoplasmic reticulum (SR) K+‐channels from wild‐type (WT) mice (where TRIC‐A is the principal subtype) and from Tric‐a knockout (KO) mice that only express TRIC‐B.We find that lone SR K+‐channels from Tric‐a KO mice have a lower open probability and gate more frequently in subconducting states than channels from WT mice but, unlike channels from WT mice, multiple channels gate with high open probability with a more than six‐fold increase in activity when four channels are present in the bilayer.No evidence was found for a direct gating interaction between ryanodine receptor and SR K+‐channels in Tric‐a KO SR, suggesting that TRIC‐B–TRIC‐B interactions are highly specific and may be important for meeting counterion requirements during excitation–contraction coupling in tissues where TRIC‐A is sparse or absent. Abstract The trimeric intracellular cation channels, TRIC‐A and TRIC‐B, represent two subtypes of sarcoplasmic reticulum (SR) K+‐channel but their individual functional roles are unknown. We therefore compared the biophysical properties of SR K+‐channels derived from the skeletal muscle of wild‐type (WT) or Tric‐a knockout (KO) mice. Because TRIC‐A is the major TRIC‐subtype in skeletal muscle, WT SR will predominantly contain TRIC‐A channels, whereas Tric‐a KO SR will only contain TRIC‐B channels. When lone SR K+‐channels were incorporated into bilayers, the open probability (Po) of channels from Tric‐a KO mice was markedly lower than that of channels from WT mice; gating was characterized by shorter opening bursts and more frequent brief subconductance openings. However, unlike channels from WT mice, the Po of SR K+‐channels from Tric‐a KO mice increased as increasing channel numbers were present in the bilayer, driving the channels into long sojourns in the fully open state. When co‐incorporated into bilayers, ryanodine receptor channels did not directly affect the gating of SR K+‐channels, nor did the presence or absence of SR K+‐channels influence ryanodine receptor activity. We suggest that because of high expression levels in striated muscle, TRIC‐A produces most of the counterion flux required during excitation‐contraction coupling. TRIC‐B, in contrast, is sparsely expressed in most cells and, although lone TRIC‐B channels exhibit low Po, the high Po levels reached by multiple TRIC‐B channels may provide a compensatory mechanism to rapidly restore K+ gradients and charge differences across the SR of tissues containing few TRIC‐A channels., Key points There are two subtypes of trimeric intracellular cation (TRIC) channels but their distinct single‐channel properties and physiological regulation have not been characterized. We examined the differences in function between native skeletal muscle sarcoplasmic reticulum (SR) K+‐channels from wild‐type (WT) mice (where TRIC‐A is the principal subtype) and from Tric‐a knockout (KO) mice that only express TRIC‐B.We find that lone SR K+‐channels from Tric‐a KO mice have a lower open probability and gate more frequently in subconducting states than channels from WT mice but, unlike channels from WT mice, multiple channels gate with high open probability with a more than six‐fold increase in activity when four channels are present in the bilayer.No evidence was found for a direct gating interaction between ryanodine receptor and SR K+‐channels in Tric‐a KO SR, suggesting that TRIC‐B–TRIC‐B interactions are highly specific and may be important for meeting counterion requirements during excitation–contraction coupling in tissues where TRIC‐A is sparse or absent.

Key points Distinct spiking patterns may arise from qualitative differences in ion channel expression (i.e. when different neurons express distinct ion channels) and/or when quantitative differences in expression levels qualitatively alter the spike generation process.We hypothesized that spiking patterns in neurons of the superficial dorsal horn (SDH) of spinal cord reflect both mechanisms.We reproduced SDH neuron spiking patterns by varying densities of KV1‐ and A‐type potassium conductances. Plotting the spiking patterns that emerge from different density combinations revealed spiking‐pattern regions separated by boundaries (bifurcations).This map suggests that certain spiking pattern combinations occur when the distribution of potassium channel densities straddle boundaries, whereas other spiking patterns reflect distinct patterns of ion channel expression. The former mechanism may explain why certain spiking patterns co‐occur in genetically identified neuron types.We also present algorithms to predict spiking pattern proportions from ion channel density distributions, and vice versa. Abstract Neurons are often classified by spiking pattern. Yet, some neurons exhibit distinct patterns under subtly different test conditions, which suggests that they operate near an abrupt transition, or bifurcation. A set of such neurons may exhibit heterogeneous spiking patterns not because of qualitative differences in which ion channels they express, but rather because quantitative differences in expression levels cause neurons to operate on opposite sides of a bifurcation. Neurons in the spinal dorsal horn, for example, respond to somatic current injection with patterns that include tonic, single, gap, delayed and reluctant spiking. It is unclear whether these patterns reflect five cell populations (defined by distinct ion channel expression patterns), heterogeneity within a single population, or some combination thereof. We reproduced all five spiking patterns in a computational model by varying the densities of a low‐threshold (KV1‐type) potassium conductance and an inactivating (A‐type) potassium conductance and found that single, gap, delayed and reluctant spiking arise when the joint probability distribution of those channel densities spans two intersecting bifurcations that divide the parameter space into quadrants, each associated with a different spiking pattern. Tonic spiking likely arises from a separate distribution of potassium channel densities. These results argue in favour of two cell populations, one characterized by tonic spiking and the other by heterogeneous spiking patterns. We present algorithms to predict spiking pattern proportions based on ion channel density distributions and, conversely, to estimate ion channel density distributions based on spiking pattern proportions. The implications for classifying cells based on spiking pattern are discussed., Key points Distinct spiking patterns may arise from qualitative differences in ion channel expression (i.e. when different neurons express distinct ion channels) and/or when quantitative differences in expression levels qualitatively alter the spike generation process.We hypothesized that spiking patterns in neurons of the superficial dorsal horn (SDH) of spinal cord reflect both mechanisms.We reproduced SDH neuron spiking patterns by varying densities of KV1‐ and A‐type potassium conductances. Plotting the spiking patterns that emerge from different density combinations revealed spiking‐pattern regions separated by boundaries (bifurcations).This map suggests that certain spiking pattern combinations occur when the distribution of potassium channel densities straddle boundaries, whereas other spiking patterns reflect distinct patterns of ion channel expression. The former mechanism may explain why certain spiking patterns co‐occur in genetically identified neuron types.We also present algorithms to predict spiking pattern proportions from ion channel density distributions, and vice versa.

PIEZO1 is a large mechanosensitive ion channel protein. Diseases associated with PIEZO1 include autosomal recessive generalised lymphatic dysplasia of Fotiou (GLDF) and autosomal dominant dehydrated hereditary stomatocytosis with or without pseudohyperkalemia and/or perinatal oedema (DHS). The two disorders show overlapping features, fetal hydrops/perinatal oedema have been reported in both. Electrophysiological studies suggest opposite mechanisms of action: the mutations identified in GLDF patients cause a loss‐of‐function mechanism of disease and mutations in DHS patients cause gain of function. This raises the question: Is the pathogenic disease mechanism behind the fetal oedema the same in the two phenotypes? In this Symposium Review, we will discuss the two conditions and highlight key questions that remain to be answered. For instance, the perinatal oedema often resolves soon after birth and we are still at a loss to understand why. Are there any mechanisms which could compensate for the faulty PIEZO1 in these patients? Are there physiological changes at birth that are less reliant on the function of PIEZO1? Thus, there is a clear need for further studies into the two disorders, in order to fully understand the role of PIEZO1 in health and disease. [Image: see text]

The tightly regulated opening and closure of ion channels underlies the electrical signals that are vital for a wide range of physiological processes. Two decades ago the first atomic level view of ion channel structures led to a detailed understanding of ion selectivity and conduction. In recent years spectacular developments in the field of cryo-electron microscopy have resulted in cryo-EM superseding crystallography as the technique of choice for determining near atomic resolution structures of ion channels. Here, we will review the recent developments in cryo-EM and its specific application to the study of ion channel gating. We will highlight the advantages and disadvantages of the current technology and where the field is likely to head in the next few years. This article is protected by copyright. All rights reserved.

The evolutionarily conserved Piezo family of proteins, including Piezo1 and Piezo2, encodes the long‐sought‐after mammalian mechanosensitive cation channels that play critical roles in various mechanotransduction processes such as touch, pain, proprioception, vascular development and blood pressure regulation. Mammalian Piezo proteins contain over 2500 amino acids with numerous predicted transmembrane segments, and do not bear sequence homology with any known class of ion channels. Thus, it is imperative, but challenging, to understand how they serve as effective mechanotransducers for converting mechanical force into electrochemical signals. Here, we review the recent major breakthroughs in determining the three‐bladed, propeller‐shaped structure of mouse Piezo1 using the state‐of‐the‐art cryo‐electron microscopy (cryo‐EM) and functionally dissecting out the molecular bases that define its ion permeation and mechanotransduction properties, which provide key insights into clarifying its oligomeric status and pore‐forming region. We also discuss the hypothesis that the complex Piezo proteins can be deduced into discrete mechanotransduction and ion‐conducting pore modules, which coordinate to fulfil their specialized function in mechanical sensing and transduction, ion permeation and selection. [Image: see text]

Distinct spiking patterns may arise from qualitative differences in ion channel expression (i.e. when different neurons express distinct ion channels) and/or when quantitative differences in expression levels qualitatively alter the spike generation process. We hypothesized that spiking patterns in neurons of the superficial dorsal horn (SDH) of spinal cord reflect both mechanisms. We reproduced SDH neuron spiking patterns by varying densities of KNeurons are often classified by spiking pattern. Yet, some neurons exhibit distinct patterns under subtly different test conditions, which suggests that they operate near an abrupt transition, or bifurcation. A set of such neurons may exhibit heterogeneous spiking patterns not because of qualitative differences in which ion channels they express, but rather because quantitative differences in expression levels cause neurons to operate on opposite sides of a bifurcation. Neurons in the spinal dorsal horn, for example, respond to somatic current injection with patterns that include tonic, single, gap, delayed and reluctant spiking. It is unclear whether these patterns reflect five cell populations (defined by distinct ion channel expression patterns), heterogeneity within a single population, or some combination thereof. We reproduced all five spiking patterns in a computational model by varying the densities of a low-threshold (K

Key points: We constructed a comprehensive mathematical model of the spontaneous electrical activity of a human sinoatrial node (SAN) pacemaker cell, starting from the recent Severi–DiFrancesco model of rabbit SAN cells. Our model is based on electrophysiological data from isolated human SAN pacemaker cells and closely matches the action potentials and calcium transient that were recorded experimentally. Simulated ion channelopathies explain the clinically observed changes in heart rate in corresponding mutation carriers, providing an independent qualitative validation of the model. The model shows that the modulatory role of the ‘funny current’ (If) in the pacing rate of human SAN pacemaker cells is highly similar to that of rabbit SAN cells, despite its considerably lower amplitude. The model may prove useful in the design of experiments and the development of heart-rate modulating drugs. Abstract: The sinoatrial node (SAN) is the normal pacemaker of the mammalian heart. Over several decades, a large amount of data on the ionic mechanisms underlying the spontaneous electrical activity of SAN pacemaker cells has been obtained, mostly in experiments on single cells isolated from rabbit SAN. This wealth of data has allowed the development of mathematical models of the electrical activity of rabbit SAN pacemaker cells. The present study aimed to construct a comprehensive model of the electrical activity of a human SAN pacemaker cell using recently obtained electrophysiological data from human SAN pacemaker cells. We based our model on the recent Severi–DiFrancesco model of a rabbit SAN pacemaker cell. The action potential and calcium transient of the resulting model are close to the experimentally recorded values. The model has a much smaller ‘funny current’ (If) than do rabbit cells, although its modulatory role is highly similar. Changes in pacing rate upon the implementation of mutations associated with sinus node dysfunction agree with the clinical observations. This agreement holds for both loss-of-function and gain-of-function mutations in the HCN4, SCN5A and KCNQ1 genes, underlying ion channelopathies in If, fast sodium current and slow delayed rectifier potassium current, respectively. We conclude that our human SAN cell model can be a useful tool in the design of experiments and the development of drugs that aim to modulate heart rate.

Key points The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric‐a gene knockout (KO) alters the single‐channel function of skeletal RyR (RyR1).We find that RyR1 from Tric‐a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+, ATP, caffeine and luminal Ca2+.In the presence of Mg2+, ATP cannot effectively activate RyR1 from Tric‐a KO mice.Additionally, RyR1 from Tric‐a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of β‐adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+‐release.The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+‐release observed in skeletal muscle from Tric‐a KO mice, further highlighting the importance of TRIC‐A for normal physiological regulation of SR Ca2+‐release in skeletal muscle. Abstract The type A trimeric intracellular cation channel (TRIC‐A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric‐a knockout (KO) mice is characterized by Ca2+ overloaded and swollen SR and by changes in the properties of SR Ca2+ release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric‐a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage‐clamp conditions. We find that RyR1 channels from Tric‐a KO mice respond normally to cytosolic Ca2+, ATP, adenine, caffeine and to luminal Ca2+. However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg2+, ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC‐A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC‐A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K+ fluxes across the SR via TRIC‐A., Key points The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric‐a gene knockout (KO) alters the single‐channel function of skeletal RyR (RyR1).We find that RyR1 from Tric‐a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+, ATP, caffeine and luminal Ca2+.In the presence of Mg2+, ATP cannot effectively activate RyR1 from Tric‐a KO mice.Additionally, RyR1 from Tric‐a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of β‐adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+‐release.The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+‐release observed in skeletal muscle from Tric‐a KO mice, further highlighting the importance of TRIC‐A for normal physiological regulation of SR Ca2+‐release in skeletal muscle.

Both uncoupling protein 1 (UCP1) and UCP3 are important for mammalian thermoregulation. UCP1 and UCP3 in brown adipose tissue mediate early and late phases of sympathomimetic thermogenesis, respectively. Lipopolysaccharide thermogenesis requires skeletal muscle UCP3 but not UCP1. Acute noradrenaline-induced hyperthermia requires UCP1 but not UCP3. Loss of both UCP1 and UCP3 accelerate the loss of body temperature compared to UCP1KO alone during acute cold exposure.Uncoupling protein 1 (UCP1) is the established mediator of brown adipose tissue-dependent thermogenesis. In contrast, the role of UCP3, expressed in both skeletal muscle and brown adipose tissue, in thermoregulatory physiology is less well understood. Here, we show that mice lacking UCP3 (UCP3KO) have impaired sympathomimetic (methamphetamine) and completely abrogated lipopolysaccharide (LPS) thermogenesis, but a normal response to noradrenaline. By comparison, UCP1 knockout (UCP1KO) mice exhibit blunted methamphetamine and fully inhibited noradrenaline thermogenesis, but an increased febrile response to LPS. We further establish that mice lacking both UCP1 and 3 (UCPDK) fail to show methamphetamine-induced hyperthermia, and have a markedly accelerated loss of body temperature and survival after cold exposure compared to UCP1KO mice. Finally, we show that skeletal muscle-specific human UCP3 expression is able to significantly rescue LPS, but not sympathomimetic thermogenesis blunted in UCP3KO mice. These studies identify UCP3 as an important mediator of physiological thermogenesis and support a renewed focus on targeting UCP3 in metabolic physiology.

Sensory neurons that innervate the bone marrow provide the CNS with information about pain associated with bone disease and pathology, but little is known of their function. Here we use a novel in vivo bone-nerve electrophysiological preparation to study how they respond to noxious mechanical stimulation delivered by increasing intra-osseous pressure. We provide evidence that sensory neurons that innervate the bone marrow respond to high threshold noxious mechanical stimulation, have response properties consistent with a role in nociception, provide information about different features of an intra-osseous pressure stimulus and express the Piezo2 mechano-transducer molecule. Our findings show how some bone marrow nociceptors signal pain in bony diseases and pathologies that involve a mechanical disturbance or increased intra-osseous pressure, and that the Piezo2 mechano-transducer may be involved.Whilst the sensory neurons and nerve terminals that innervate bone marrow have a morphology and molecular phenotype consistent with a role in nociception, little is known about their physiology or the mechanisms that generate and maintain bone pain. In the present study, we provide evidence that Aδ nociceptors that innervate the bone marrow respond to high threshold noxious mechanical stimulation, exhibit fatigue in response to prior stimulation and in some cases can be sensitized by capsaicin. They can be classified on the basis of their response properties as either phasic-tonic units that appear to code for different intensities of intra-osseous pressure, or phasic units that code for the rate of change in intra-osseous pressure. Three different subclasses of mechanically sensitive Aδ units were observed: phasic units that were sensitized by capsaicin, phasic units that were not sensitized by capsaicin and phasic-tonic units (that were not sensitized by capsaicin). These could also, in part, be distinguished by differences in their thresholds for activation, mean discharge frequency, latency to peak activation and peak-to-peak action potential amplitude. The majority of small-diameter myelinated sensory neurons projecting to the bone marrow expressed Piezo2. Our findings indicate that Aδ mechano-nociceptors are likely to play an important role in generating and maintaining pain in response to bony pathologies that involve a mechanical disturbance or increased intra-osseous pressure, and imply that Piezo2 signalling may be involved in mechano-transduction in these receptors.

Chronic atrial fibrillation (AF) is associated with structural and electrical remodelling in the atria, which are associated with a high recurrence of AF. Through biophysically detailed computer modelling, this study investigated mechanisms by which AF-induced electrical remodelling promotes and perpetuates AF. A family of Courtemanche-Ramirez-Nattel variant models of human atrial cell action potentials (APs), taking into account of intrinsic atrial electrophysiological properties, was modified to incorporate various experimental data sets on AF-induced changes of major ionic channel currents (ICaL, IKur, Ito, IK1, IKs, INaCa) and on intracellular Ca(2+) handling. The single cell models for control and AF-remodelled conditions were incorporated into multicellular three-dimensional (3D) atrial tissue models. Effects of the AF-induced electrical remodelling were quantified as the changes of AP profile, AP duration (APD) and its dispersion across the atria, and the vulnerability of atrial tissue to the initiation of re-entry. The dynamic behaviour of re-entrant excitation waves in the 3D models was characterised. In our simulations, AF-induced electrical remodelling abbreviated atrial APD non-uniformly across the atria; this resulted in relatively short APDs co-existing with marked regional differences in the APD at junctions of the crista terminalis/pectinate muscle, pulmonary veins/left atrium. As a result, the measured tissue vulnerability to re-entry initiation at these tissue junctions was increased. The AF-induced electrical remodelling also stabilized and accelerated re-entrant excitation waves, leading to rapid and sustained re-entry. Under the AF-remodelled condition, re-entrant scroll waves in the 3D model degenerated into persistent and erratic wavelets, leading to fibrillation. In conclusion, realistic 3D atrial tissue models indicate that AF-induced electrical remodelling produces regionally heterogeneous and shortened APD; these respectively facilitate initiation and maintenance of re-entrant excitation waves.

Mechanisms underlying fatigue development and limitations for performance during intense exercise have been intensively studied during the past couple of decades. Fatigue development may involve several interacting factors and depends on type of exercise undertaken and training level of the individual. Intense exercise (½–6 min) causes major ionic perturbations (Ca2+, Cl−, H+, K+, lactate− and Na+) that may reduce sarcolemmal excitability, Ca2+ release and force production of skeletal muscle. Maintenance of ion homeostasis is thus essential to sustain force production and power output during intense exercise. Regular speed endurance training (SET), i.e. exercise performed at intensities above that corresponding to maximum oxygen consumption (V˙O2, max ), enhances intense exercise performance. However, most of the studies that have provided mechanistic insight into the beneficial effects of SET have been conducted in untrained and recreationally active individuals, making extrapolation towards athletes’ performance difficult. Nevertheless, recent studies indicate that only a few weeks of SET enhances intense exercise performance in highly trained individuals. In these studies, the enhanced performance was not associated with changes in V˙O2, max and muscle oxidative capacity, but rather with adaptations in muscle ion handling, including lowered interstitial concentrations of K+ during and in recovery from intense exercise, improved lactate−–H+ transport and H+ regulation, and enhanced Ca2+ release function. The purpose of this Topical Review is to provide an overview of the effect of SET and to discuss potential mechanisms underlying enhancements in performance induced by SET in already well‐trained individuals with special emphasis on ion handling in skeletal muscle.

The hyperpolarization-activated current (I(h)) has been implicated in nociception/pain, but its expression levels in nociceptors remained unknown. We recorded I(h) magnitude and properties by voltage clamp from dorsal root ganglion (DRG) neurons in vivo, after classifying them as nociceptive or low-threshold-mechanoreceptors (LTMs) and as having C-, Aδ- or Aα/β-conduction velocities (CVs). For both nociceptors andLTMs, I(h) amplitude and I(h) density (at -100 mV) were significantly positively correlated with CV.Median I(h) magnitudes and I(h) density in neuronal subgroupswere respectively:muscle spindle afferents(MSAs):-4.6 nA,-33 pA pF(-1); cutaneous Aα/β LTMs: -2.2 nA, -20 pA pF(-1); Aβ-nociceptors: -2.6 nA, -21 pA pF(-1); both Aδ-LTMs and nociceptors: -1.3 nA, ∼-14 pA pF(-1); C-LTMs: -0.4 nA, -7.6 pA pF(-1); and C-nociceptors: -0.26 nA, -5 pApF(-1). I(h) activation slow time constants (slow τ values) were strongly correlated with fast τ values; both were shortest in MSAs. Most neurons had τ values consistent with HCN1-related I(h); others had τ values closer to HCN1+HCN2 channels, or HCN2 in the presence of cAMP. In contrast, median half-activation voltages (V(0.5)) of -80 to -86 mV for neuronal subgroups suggest contributions of HCN2 to I(h). τ values were unrelated to CV but were inversely correlated with I(h) and I(h) density for all non-MSA LTMs, and for Aδ-nociceptors. From activation curves ∼2-7% of I(h)would be activated at normal membrane potentials. The high I(h) may be important for excitability of A-nociceptors (responsible for sharp/pricking-type pain) and Aα/β-LTMs (tactile sensations and proprioception). Underlying HCN expression in these subgroups therefore needs to be determined. Altered high I(h) may be important for excitability of A-nociceptors (responsible for sharp/pricking-type pain) and Aα/β-LTMs (tactile sensations and proprioception). Underlying HCN expression in these subgroups therefore needs to be determined. Altered Ih expression and/or properties (e.g. in chronic/pathological pain states) may influence both nociceptor and LTM excitability.expression and/or properties (e.g. in chronic/pathological pain states) may influence both nociceptor and LTM excitability.

Carotid body (CB) glomus cells mediate acute oxygen sensing and the initiation of the hypoxic ventilatory response, yet the gene expression profile of these cells is not available. We demonstrate that the single cell RNA-Seq method is a powerful tool for identifying highly expressed genes in CB glomus cells. Our single cell RNA-Seq results characterized novel CB glomus cell genes, including members of the G protein-coupled receptor signalling pathway, ion channels and atypical mitochondrial electron transport chain subunits. A heterologous cell-based screening identified acetate (which is known to affect CB glomus cell activity) as an agonist for the most highly abundant G protein-coupled receptor (Olfr78) in CB glomus cells. These data established the first transcriptome profile of CB glomus cells, highlighting genes with potential implications in CB chemosensory function.The carotid body (CB) is a major arterial chemoreceptor containing glomus cells whose activities are regulated by changes in arterial blood content, including oxygen. Despite significant advancements in the characterization of their physiological properties, our understanding of the underlying molecular machinery and signalling pathway in CB glomus cells is still limited. To overcome this, we employed the single cell RNA-Seq method by performing next-generation sequencing on single glomus cell-derived cDNAs to eliminate contamination of genes derived from other cell types present in the CB. Using this method, we identified a set of genes abundantly expressed in glomus cells, which contained novel glomus cell-specific genes. Transcriptome and subsequent in situ hybridization and immunohistochemistry analyses identified abundant G protein-coupled receptor signalling pathway components and various types of ion channels, as well as members of the hypoxia-inducible factors pathway. A short-chain fatty acid olfactory receptor Olfr78, recently implicated in CB function, was the most abundant G protein-coupled receptor. Two atypical mitochondrial electron transport chain subunits (Ndufa4l2 and Cox4i2) were among the most specifically expressed genes in CB glomus cells, highlighting their potential roles in mitochondria-mediated oxygen sensing. The wealth of information provided by the present study offers a valuable foundation for identifying molecules functioning in the CB.

Rheumatoid arthritis (RA) is a progressive disease that affects both pediatric and adult populations. The cellular basis for RA has been investigated extensively using animal models, human tissues and isolated cells in culture. However, many aspects of its aetiology and molecular mechanisms remain unknown. Some of the electrophysiological principles that regulate secretion of essential lubricants (hyaluronan and lubricin) and cytokines from synovial fibroblasts have been identified. Data sets describing the main types of ion channels that are expressed in human synovial fibroblast preparations have begun to provide important new insights into the interplay among: (i) ion fluxes, (ii) Ca2+ release from the endoplasmic reticulum, (iii) intercellular coupling, and (iv) both transient and longer duration changes in synovial fibroblast membrane potential. A combination of this information, knowledge of similar patterns of responses in cells that regulate the immune system, and the availability of adult human synovial fibroblasts are likely to provide new pathophysiological insights.

Fragile X‐associated disorders are a family of genetic conditions resulting from the partial or complete loss of fragile X mental retardation protein (FMRP). Among these disorders is fragile X syndrome, the most common cause of inherited intellectual disability and autism. FMRP is an RNA‐binding protein involved in the control of local translation, which has pleiotropic effects, in particular on synaptic function. Analysis of the brain FMRP transcriptome has revealed hundreds of potential mRNA targets encoding postsynaptic and presynaptic proteins, including a number of ion channels. FMRP has been confirmed to bind voltage‐gated potassium channels (Kv3.1 and Kv4.2) mRNAs and regulates their expression in somatodendritic compartments of neurons. Recent studies have uncovered a number of additional roles for FMRP besides RNA regulation. FMRP was shown to directly interact with, and modulate, a number of ion channel complexes. The sodium‐activated potassium (Slack) channel was the first ion channel shown to directly interact with FMRP; this interaction alters the single‐channel properties of the Slack channel. FMRP was also shown to interact with the auxiliary β4 subunit of the calcium‐activated potassium (BK) channel; this interaction increases calcium‐dependent activation of the BK channel. More recently, FMRP was shown to directly interact with the voltage‐gated calcium channel, Cav2.2, and reduce its trafficking to the plasma membrane. Studies performed on animal models of fragile X syndrome have revealed links between modifications of ion channel activity and changes in neuronal excitability, suggesting that these modifications could contribute to the phenotypes observed in patients with fragile X‐associated disorders.

White to brown adipose tissue conversion and thermogenesis can be ignited by different conditions or agents and its sustainability over the long term is still unclear. Browning of rat retroperitoneal white adipose tissue (rpWAT) during cold acclimation involves two temporally apparent components: (1) a predominant non-selective browning of most adipocytes and an initial sharp but transient induction of uncoupling protein 1, peroxisome proliferator-activated receptor (PPAR) coactivator-1α, PPARγ and PPARα expression, and (2) the subsistence of relatively few thermogenically competent adipocytes after 45 days of cold acclimation. The different behaviours of two rpWAT beige/brown adipocyte subsets control temporal aspects of the browning process, and thus regulation of both components may influence body weight and the potential successfulness of anti-obesity therapies.Conversion of white into brown adipose tissue may have important implications in obesity resistance and treatment. Several browning agents or conditions ignite thermogenesis in white adipose tissue (WAT). To reveal the capacity of WAT to function in a brownish/burning mode over the long term, we investigated the progression of the rat retroperitoneal WAT (rpWAT) browning during 45 days of cold acclimation. During the early stages of cold acclimation, the majority of rpWAT adipocytes underwent multilocularization and thermogenic-profile induction, as demonstrated by the presence of a multitude of uncoupling protein 1 (UCP1)-immunopositive paucilocular adipocytes containing peroxisome proliferator-activated receptor (PPAR) coactivator-1α (PGC-1α) and PR domain-containing 16 (PRDM16) in their nuclei. After 45 days, all adipocytes remained PRDM16 immunopositive, but only a few multilocular adipocytes rich in mitochondria remained UCP1/PGC-1α immunopositive. Molecular evidence showed that thermogenic recruitment of rpWAT occurred following cold exposure, but returned to starting levels after cold acclimation. Compared with controls (22 ± 1 °C), levels of UCP1 mRNA increased in parallel with PPARγ (PPARα from days 1 to 7 and PGC-1α on day 1). Transcriptional recruitment of rpWAT was followed by an increase in UCP1 protein content (from days 1 to 21). Results clearly showed that most of the adipocytes within rpWAT underwent transient brown-fat-like thermogenic recruitment upon stimulation, but only a minority of cells retained a brown adipose tissue-like phenotype after the attainment of cold acclimation. Therefore, browning of WAT is dependent on both maintaining the thermogenic response and retaining enough brown-like thermogenically competent adipocytes in the long-term. Both aspects of browning could be important for long-term energy homeostasis and body-weight regulation.

Hyperpolarization-activated inward current (If) and changes in the messenger RNA (mRNA) expression levels of hyperpolarization-activated cyclic nucleotide-gated channel (HCN)2 and HCN4 encoding If channels of the rat heart were studied in control and hypertrophied myocytes isolated from three ventricular regions: the septum (S), the left ventricular free wall (LV) and the right ventricular free wall (RV). Electrophysiological experiments were conducted by ruptured and perforated-patch clamp techniques and quantification of mRNA levels was carried out by quantitative reverse transcriptase polymerase chain reaction. The occurrence, density and maximal specific conductance of If were found to be significantly higher in hypertrophied ventricular myocytes isolated from S and LV than in those isolated from RV or sham-operated rats. Half-maximal activation potential, the slope of the activation curve and the threshold for activation were similar in ventricular myocytes from sham and aortic stenosed rats in the three regions studied. Isoproterenol 1 micromol l-1 increased current size by shifting current activation to more positive potentials in both sham and hypertrophied myocytes. When we studied the mRNA levels of If channel isoforms present in the ventricle, we found a significant increase of HCN2 and HCN4 mRNA levels in hypertrophied myocytes from S and LV but not in RV. We conclude that the occurrence, density and conductance of If is higher in hypertrophied than in control ventricular myocytes, S being the region where all these changes were most evident. These findings are associated with a higher expression of HCN2 and HCN4 mRNA levels in the two regions that developed hypertrophy., This study was supported by the Ministry of Science and Technology of Spain (SAF99-0068 and BFI2002-00536).

The mammalian cortex is densely populated by extensively branching, thin, unmyelinated axons that form en passant synapses. Some thin axons in the peripheral nervous system hyperpolarize if action potential frequency exceeds 1-5 Hz. To test the hypothesis that cortical axons also show activity-induced hyperpolarization, we recorded extracellularly from individual CA3 pyramidal neurons while activating their axon with trains consisting of 30 electrical stimuli. Synaptic excitation was blocked by kynurenic acid. We observed a positive correlation between stimulation strength and the number of consecutive axonal stimuli that resulted in soma spikes, suggesting that the threshold increased as a function of the number of spikes. During trains without response failures there was always a cumulative increase in the soma response latency. Intermittent failures, however, decreased the latency of the subsequent response. At frequencies of1 Hz, the threshold and latency increases were enhanced by blocking the hyperpolarization-activated H current (Ih)by applying the specific Ih blocker ZD7288 (25 microM) or 2 mM Cs+. Under these conditions, response failures occurred after 15-25 stimuli, independent of the stimulation strength. Adding GABA receptor blockers (saclofen and bicuculline) and a blocker of metabotropic glutamate receptors did not change the activity-induced latency increase in recordings of the compound action potential. We interpret these results as an activity-induced hyperpolarization that is partly counteracted by Ih. Such a hyperpolarization may influence transmitter release and the conduction reliability of these axons.

The circumventricular organs (CVOs) are ideal locations at which circulating pyrogens may act to communicate with the CNS during an immune challenge. Their dense vasculature and fenestrated capillaries allow direct access of these pyrogens to CNS tissue without impediment of the blood-brain barrier (BBB). One such CVO, the subfornical organ (SFO), has been implicated as a site at which the circulating endogenous pyrogen interleukin 1beta (IL-1beta) acts to initiate the febrile response. This study was designed to determine the response of rat SFO neurons to IL-1beta (1 nM to 100 fM) using whole-cell current-clamp and voltage-damp techniques. We found that physiological(subseptic) concentrations of IL-1beta (1 pM, 500 fM, 100 fm) induced a transient depolarization in SFO neurons accompanied by a significant increase in spike frequency. In contrast,pharmacological (septic) concentrations of IL-1beta (1 nM) evoked a sustained hyperpolarization. While depolarizations in response to IL-1beta were abolished by treatment of cells with the IL-1 receptor antagonist (IL-1ra), hyperpolarizations were still observed. Voltage-clamp analysis revealed that the majority (85 %) of SFO neurons responding to IL-1beta with depolarization (29 of 34 cells) exhibited an electrophysiological profile characterized by a dominant delayed rectifier potassium current (DIK), a conductance that we also found to be reduced to 84.4 +/- 3.3 % of control by bath application of IL-1beta. In addition, using slow voltage ramps we demonstrated that IL-1beta activates a non-selective cationic current (INSC) with a reversal potential of -38.8 +/- 1.8 mV. These studies identify the cellular mechanisms through which IL-1beta can influence the excitability of SFO neurons and, as a consequence of such actions, initiate the febrile response to exogenous pyrogens.

Electrophysiological recording was used to study a type of slow excitatory postsynaptic potential (slow EPSP) that was mediated by release of ATP and its action at P2Y1 receptors on morphologically identified neurones in the submucosal plexus of guinea-pig small intestine. MRS2179, a selective P2Y1 purinergic receptor antagonist, blocked both the slow EPSP and mimicry of the EPSP by exogenously applied ATP. Increased conductance accounted for the depolarization phase of the EPSP, which occurred exclusively in neurones with S-type electrophysiological behaviour and uniaxonal morphology. The purinergic excitatory input to the submucosal neurones came from neighbouring neurones in the same plexus, from neurones in the myenteric plexus and from sympathetic postganglionic neurones. ATP-mediated EPSPs occurred coincident with fast nicotinic synaptic potentials evoked by the myenteric projections and with noradrenergic IPSPs evoked by sympathetic fibres that innervated the same neurones. The P2Y1 receptor on the neurones was identified as a metabotropic receptor linked to activation of phospholipase C, synthesis of inositol 1,4,5-trisphosphate and mobilization of Ca2+ from intracellular stores.

At certain synapses in the brain, Ca2+-permeable AMPA receptor (AMPAR) channels represent an important pathway for synaptically controlled Ca2+ entry. However, the molecular determinants of this Ca2+ influx are poorly defined. In NMDA receptor (NMDAR) channels, where the influx is much greater, the extracellular vestibule, specifically the M3 segment and regions C-terminal to it in the NR1 subunit, contains elements critical to their high Ca2+ influx under physiological conditions. We therefore investigated the contribution of homologous positions in AMPAR as well as kainate receptor (KAR) subunits to the process of Ca2+ influx. Substitutions of a conserved asparagine (N) in M3 of AMPAR GluR-B(Q) channels strongly attenuated Ca2+ permeability measured using reversal potentials under biionic conditions and fractional Ca2+ currents recorded under physiological conditions. Hence, as in NMDAR channels, the conserved N makes a significant contribution to Ca2+ influx in AMPAR channels. In addition, C-terminal to M3, substitutions of negatively (glutamate, E) or positively (arginine, R) charged residues also altered Ca2+ influx. However, in contrast to charged residues occupying homologous positions in NMDAR channels, these effects were about equal and opposite suggesting that this ER in AMPARs does not contribute significantly to the mechanism of Ca2+ influx. Opposite charge substitutions of two negative residues C-terminal to M3 in KAR GluR-6(Q) subunits had no effect on Ca2+ permeability. We conclude that the different contribution of residues C-terminal to M3 to Ca2+ permeation in NMDAR and non-NMDAR channels reflects a different positioning of these residues relative to the tip of the M2 loop.

Opening of the mitochondrial permeability transition pore (MPTP) is thought to be a critical event in mediating the damage to hearts that accompanies their reperfusion following prolonged ischaemia. Protection from reperfusion injury occurs if the prolonged ischaemic period is preceded by short ischaemic periods followed by recovery. Here we investigate whether such ischaemic preconditioning (IPC) is accompanied by inhibition of MPTP opening. MPTP opening in Langendorff-perfused rat hearts was determined by perfusion with 2-deoxy[3H]glucose ([3H]DOG) and measurement of mitochondrial [3H]DOG entrapment. We demonstrate that IPC inhibits initial MPTP opening in hearts reperfused after 30 min global ischaemia, and subsequently enhances pore closure as hearts recover. However, MPTP opening in mitochondria isolated from IPC hearts occurred more readily than control mitochondria, implying that MPTP inhibition by IPC in situ was secondary to other factors such as decreased calcium overload and oxidative stress. Hearts perfused with cyclosporin A or sanglifehrin A, powerful inhibitors of the MPTP, also recovered better from ischaemia than controls (improved haemodynamic function and less lactate dehydrogenase release). However, the mitochondrial DOG entrapment technique showed these agents to be less effective than IPC at preventing MPTP opening. Our data suggest that protection from reperfusion injury is better achieved by reducing factors that induce MPTP opening than by inhibiting the MPTP directly.

Bipolar sensory neurons within the vomeronasal organ (VNO) are thought to mediate the detection of pheromones in vertebrates. In the mouse, VNO neurons respond to pheromones with a rise in intracellular Ca2+ that accompanies a depolarization of the cell. Transduction of the pheromone appears to occur through the activation of a phosphatidylinositol signalling pathway, but the ion channels that respond to this signalling pathway have not been identified. In this report patch-clamp recording from hamster vomeronasal sensory neurons was used to identify second-messenger-gated channels that might play a role in transduction. The results demonstrate that VNO neurons show abundant expression of a Ca2+-activated non-selective (CaNS) cation channel. The CaNS channel does not discriminate between Na+ and K+ and has a slope conductance of 22 pS. Half-activation of the channel occurs at a Ca2+ concentration of 0.5 mM (at -80 mV). The probability of opening (Po) of the channel is further augmented at positive potentials, and shows an e-fold voltage dependence per 37 mV. The channel exhibits rapid rundown following patch excision with Po decreasing from near 1.0 to near 0. The adenine nucleotides ATP and cAMP block the channel with an apparent affinity of 3 and 42 microM, respectively (-80 mV). Both the activation of the channel by Ca2+ and the block of the channel by adenine nucleotides show a mild voltage dependence, which can be accounted for by the voltage dependence of Po. The properties of this channel make it a candidate to either directly mediate vomeronasal sensory transduction, or to amplify the primary sensory response.

An important feature of ligand-gated ion channels is their exquisite ability to discriminate between ions. Still, little is known about the mechanisms underlying, or structural determinates of, this ability. We examined the structural elements underlying the ionic selectivity of rho1 GABA receptors expressed in Xenopus oocytes and human embryonic kidney cells using site-directed mutagenesis and two-electrode voltage-clamp or patch-clamp techniques. The wild-type GABA receptor was chloride selective, with a small but significant permeability to potassium (PNa+ : PK+ : PCl- = 0 : 0.03 :1). Mutation of an alanine to glutamate at position 291 (thought to be located at the intracellular end of the second transmembrane domain), formed a channel that exhibited little discrimination among ions (0.70:0.87:1), while deletion of a neighbouring proline (290) was chloride selective, but had elevated cation permeabilities compared to the wild-type channel (0.12 : 0.14 : 1). Together, the two mutations (DeltaP290/A291E) caused a reversal of selectivity (2.72 : 3.59 : 1). We also examined the effects of neutralizing and reversing the charge of the adjacent, and highly conserved, arginine. Mutation of the neighbouring arginine to glutamate (R292E) increased the cation permeability similar to the DeltaP290/A291E double mutant (2.4 : 3.0 : 1), whereas neutral mutations at this position (R292M or R292C) retained chloride selectivity (0 : 0.11 : 1.0 and 0 : 0.14 : 1.0, respectively). Our experiments suggest that the effective charge near the presumed intracellular mouth of the pore is critical for ionic selectivity.

The electrical and synaptic properties of myenteric neurones in normal and inflamed guinea-pig distal colons were evaluated by intracellular microelectrode recording. Chronic inflammation was established 6 days following administration of trinitrobenzene sulfonic acid (TNBS). In S neurones, inflammation only altered synaptic inputs as the amplitude of fast excitatory postsynaptic potentials were significantly larger (31 +/- 2 mV compared to 20 +/- 1 mV) and they were more likely to receive slow excitatory synaptic input (85% compared to 55%). AH neurones displayed altered electrical properties in colitis compared to control tissues: they generated more action potentials during a maximal depolarising current pulse (7 +/- 1 compared to 1.6 +/- 0.2); they had a smaller after hyperpolarisation (9 +/- 2 mV s compared to 20 +/- 2 mV s); and they were more likely to receive fast excitatory synaptic input (74% compared to 17%), possess spontaneous activity (46% compared to 3%), and generate anodal break action potentials (58% compared to 19%). Although the resting membrane potential, input resistance and action potential characteristics were unaltered in AH neurones from inflamed tissues, they exhibited an enhanced Cs+-sensitive rectification of the current-voltage relationship. This suggests that the increase in excitability of AH neurones may involve a colitis-induced augmentation of the hyperpolarisation-activated cation current (Ih) in these cells. An increased excitability, selectively in AH neurones, suggests that the afferent limb of intrinsic motor reflexes is disrupted in the inflamed colon and this may contribute to dysmotility associated with inflammatory diseases.

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels modulate the firing rates of neuronal and cardiac pacemaker cells. HCN channels resemble voltage-gated K+ channels structurally, but much less is known about their structure-function correlation. Although modulation of K+ channel gating by external K+ is a well-known phenomenon, such a link has not been established for HCN channels. Here we examined the effects of external permeant (K+, Na+ and Li+) and non-permeant (NMG+) ions on HCN1 and HCN2 gating. Substituting 64 of 96 mM external K+ with Na+, Li+ or NMG+ positively shifted steady-state activation (∼13 mV), and preferentially slowed activation of HCN1. Mutating the pore variant C-terminal to the GYG motif in HCN1, A352, to the analogous conserved Asp in K+ channels or Arg in HCN2 produced a significant hyperpolarizing activation shift (by 5-15 mV), slowed gating kinetics (up to 6-fold), and abolished or attenuated gating responses to external K+. Whereas Na+, Li+ and NMG+ substitutions produced depolarizing activation shifts of HCN2 similar to those of HCN1, deactivation but not activation of HCN2 was exclusively decelerated. We conclude that gating and permeation of HCN channels are coupled, and that modulation of this 'pore-to-gate' coupling by external K+ is isoform-specific., link_to_subscribed_fulltext

The noradrenaline (NA)-induced cation current was investigated in neurones freshly isolated from rat cardiac parasympathetic ganglia using the nystatin-perforated patch recording configuration. Under current-clamp conditions, NA depolarized the membrane, eliciting repetitive action potentials. NA evoked an inward cation current under voltage-clamp conditions at a holding potential of -60 mV. The NA-induced current was inhibited by extracellular Ca2+ or Mg2+, with a half-maximal concentration of 13 microM for Ca2+ and 1.2 mM for Mg2+. Cirazoline mimicked the NA response, and prazosin and WB-4101 inhibited the NA-induced current, suggesting the contribution of an alpha1-adrenoceptor. The NA-induced current was inhibited by U73122, a phospholipase C (PLC) inhibitor. The membrane-permeable IP3 receptor blocker xestospongin-C also blocked the NA-induced current. Furthermore, pretreatment with thapsigargin and BAPTA-AM could inhibit the NA response while KN-62, phorbol 12-myristate 13-acetate (PMA) and staurosporine had no effect. These results suggest that NA activates the extracellular Ca2+- and Mg2+-sensitive cation channels via alpha 1-adrenoceptors in neurones freshly isolated from rat cardiac parasympathetic ganglia. This activation mechanism also involves phosphoinositide breakdown, release of Ca2+ from intracellular Ca2+ stores and calmodulin. The cation channels activated by NA may play an important role in neuronal membrane depolarization in rat cardiac ganglia.

Previous studies have shown that alpha2 adrenoceptor (alpha2AR) agonists inhibit electrolyte secretion in colonic epithelia, but little is known about the molecular mechanisms involved in this process. In this study we examined the effect of alpha2AR activation on transepithelial anion secretion across isolated murine colonic epithelium. We found that alpha2AR agonists, UK 14,304, clonidine and medetomidine were potent inhibitors of anion secretion, especially in the proximal colon. Short circuit current measurements (Isc) in colonic epithelia from normal and cystic fibrosis (CF) mice showed that alpha2AR agonists inhibited basal cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion but had no effect on CFTR activation by cAMP-dependent phosphorylation. Apical administration of an ionophore, nystatin (90 microg ml-1), was used to investigate the effect of UK 14,304 on basolateral K+ transport. The Na+-K+-ATPase current, measured as ouabain-sensitive current in the absence of ion gradients, was unaltered by pretreatment of the tissue with UK 14,304 (1 microM). In the presence of a basolaterally directed K+ gradient, UK 14,304 significantly reduced nystatin-activated Isc indicating that activation of alpha2ARs inhibits basolateral K+ channels. Studies with selective K+ channel inhibitors and openers showed that alpha2AR agonists inhibited KATP channels that were tonically active in mouse colonic epithelia. RT-PCR and pharmacological studies suggested that these channels could be similar to vascular smooth muscle KATP channels comprising Kir6.1/SUR2B or Kir6.2/SUR2B subunits. Inhibition of anion secretion by alpha2AR agonists required activation of pertussis toxin-sensitive Gi/o proteins, but did not involve classical second messengers, such as cAMP or Ca2+. In summary, alpha2ARs inhibit anion secretion in colonic epithelia by acting on basolateral KATP channels, through a process that does not involve classical second messengers.

Strong electric pulses produce reversible or irreversible membrane breakdown (electroporation). We analysed the permeation properties of minute pores caused by hyperpolarization or lysophosphatidylcholine (LPC) by comparing the amount of charge carried by irregular inward currents (I(hi)) with changes in ethidium bromide (EB) fluorescence in isolated rabbit ventricular myocytes. Forty-second negative pulses from a holding potential of -20 mV induced I(hi) whose conductance increased with hyperpolarization; the mean conductance (G(hi)) was 63.6 +/- 9.9 pS pF(-1) (mean +/- S.E.M., n = 9) at -160 mV. EB fluorescence increased during voltage pulses in parallel with the time integral of I(hi) (Q(hi)), with the magnitude of the increases in nuclear EB fluorescence being 5.3 times greater than in the cytoplasm at -160 mV. Similar hyperpolarization-induced parallel increases in I(hi) and EB fluorescence were also obtained in Na(+)-free, N-methyl-D-glucamine (NMDG) solution. LPC (10 microM) induced large (101.2 +/- 21.2 pS pF(-1), n = 16), rapid (rise times, 1-10 ms) I(hi) with slow relaxation rates at -80 mV that reflected increases in G(hi) to 94.3 +/- 24.8 pS pF(-1) (n = 8) at 6 min. Plots of EB fluorescence vs. Q(hi) were well fitted by a common Hill's equation with a Hill coefficient of 0.97. Taken together, our findings indicate that hyperpolarization and LPC produced pores having the same filter properties for the permeation of small ions, including ethidium(+), and that I(hi) (carried in part by Ca(2+)) generated by membrane breakdown are capable of supplying sufficient ions to evoke abnormal excitation and contraction in cardiac myocytes.

Depolarizing afterpotentials (DAPs) that follow action potentials in magnocellular neurosecretory cells (MNCs) are thought to underlie the generation of phasic firing, a pattern that optimizes vasopressin release from the neurohypophysis. Previous work has suggested that the DAP may result from the Ca(2+)-dependent reduction of a resting K(+) conductance. Here we examined the effects of flufenamic acid (FFA), a blocker of Ca(2+)-dependent non-selective cation (CAN) channels, on DAPs and phasic firing using intracellular recordings from supraoptic MNCs in superfused explants of rat hypothalamus. Application of FFA, but not solvent (0.1 % DMSO), reversibly inhibited (IC(50) = 13.8 microM; R = 0.97) DAPs and phasic firing with a similar time course, but had no significant effects (P0.05) on membrane potential, spike threshold and input resistance, nor on the frequency and amplitude of spontaneous synaptic potentials. Moreover, FFA did not affect (P0.05) the amplitude, duration, undershoot, or frequency-dependent broadening of action potentials elicited during the spike trains used to evoke DAPs. These findings suggest that FFA inhibits the DAP by directly blocking the channels responsible for its production, rather than by interfering with Ca(2+) influx. They also support a role for DAPs in the generation of phasic firing in MNCs. Finally, the absence of a depolarization and increased membrane resistance upon application of FFA suggests that the DAP in MNCs may not be due to the inhibition of resting K(+) current, but to the activation of CAN channels.

Skin temperature is sensed by peripheral thermoreceptors. Using the neuronal soma in primary culture as a model of the receptor terminal, we have investigated the mechanisms of cold transduction in thermoreceptive neurones from rat dorsal root ganglia. Cold-sensitive neurones were pre-selected by screening for an increase in [Ca(2+)](i) on cooling; 49 % of them were also excited by 0.5 microM capsaicin. Action potentials and voltage-gated currents of cold-sensitive neurones were clearly distinct from those of cold-insensitive neurones. All cold-sensitive neurones expressed an inward current activated by cold and sensitised by (-)-menthol, which was absent from cold-insensitive neurones. This current was carried mainly by Na(+) ions and caused a depolarisation on cooling accompanied by action potentials, inducing voltage-gated Ca(2+) entry; a minor fraction of Ca(2+) entry was voltage-independent. Application of (-)-menthol shifted the threshold temperatures of the cold-induced depolarisation and the inward current to the same extent, indicating that the cold- and menthol-activated current normally sets the threshold temperature for depolarisation during cooling. The action of menthol was stereospecific, with the (+)-isomer being a less effective agonist than the (-)-isomer. Extracellular Ca(2+) modulated the cold- and menthol-activated current in a similar way to its action on intact cold receptors: lowered [Ca(2+)](o) sensitised the current, while raised [Ca(2+)](o) antagonised the menthol-induced sensitisation. During long cooling pulses the current showed adaptation, which depended on extracellular Ca(2+) and was mediated by a rise in [Ca(2+)](i). This adaptation consisted of a shift in the temperature sensitivity of the channel. In capsaicin-sensitive neurones, capsaicin application caused a profound depression of the cold-activated current. Inclusion of nerve growth factor in the culture medium shifted the threshold of the cold-activated current towards warmer temperatures. The current was blocked by 50 microM capsazepine and 100 microM SKF 96365. We conclude that the cold- and menthol-activated current is the major mechanism responsible for cold-induced depolarisation in DRG neurones, and largely accounts for the known transduction properties of intact cold receptors.

Coherent network oscillations in the brain are correlated with different behavioural states. Intrinsic resonance properties of neurons provide a basis for such oscillations. In the hippocampus, CA1 pyramidal neurons show resonance at theta (theta) frequencies (2-7 Hz). To study the mechanisms underlying theta-resonance, we performed whole-cell recordings from CA1 pyramidal cells (n = 73) in rat hippocampal slices. Oscillating current injections at different frequencies (ZAP protocol), revealed clear resonance with peak impedance at 2-5 Hz at approximately 33 degrees C (increasing to approximately 7 Hz at approximately 38 degrees C). The theta-resonance showed a U-shaped voltage dependence, being strong at subthreshold, depolarized (approximately -60 mV) and hyperpolarized (approximately -80 mV) potentials, but weaker near the resting potential (-72 mV). Voltage clamp experiments revealed three non-inactivating currents operating in the subthreshold voltage range: (1) M-current (I(M)), which activated positive to -65 mV and was blocked by the M/KCNQ channel blocker XE991 (10 microM); (2) h-current (I(h)), which activated negative to -65 mV and was blocked by the h/HCN channel blocker ZD7288 (10 microM); and (3) a persistent Na(+) current (I(NaP)), which activated positive to -65 mV and was blocked by tetrodotoxin (TTX, 1 microM). In current clamp, XE991 or TTX suppressed the resonance at depolarized, but not hyperpolarized membrane potentials, whereas ZD7288 abolished the resonance only at hyperpolarized potentials. We conclude that these cells show two forms of theta-resonance: "M-resonance" generated by the M-current and persistent Na(+) current in depolarized cells, and "H-resonance" generated by the h-current in hyperpolarized cells. Computer simulations supported this interpretation. These results suggest a novel function for M/KCNQ channels in the brain: to facilitate neuronal resonance and network oscillations in cortical neurons, thus providing a basis for an oscillation-based neural code.

Spontaneous, rhythmical contractions, or vasomotion, can be recorded from cerebral vessels under both normal physiological and pathophysiological conditions. Using electrophysiology to study changes in membrane potential, the ratiometric calcium indicator Fura-2 AM to study changes in [Ca(2+)](i) in both the arterial wall and in individual smooth muscle cells (SMCs), and video microscopy to study changes in vessel diameter, we have investigated the cellular mechanisms underlying vasomotion in the juvenile rat basilar artery. During vasomotion, rhythmical oscillations in both membrane potential and [Ca(2+)](i) were found to precede rhythmical contractions. Nifedipine depolarized SMCs and abolished rhythmical contractions and depolarizations. [Ca(2+)](i) oscillations in the arterial wall became reduced and irregular, while [Ca(2+)](i) oscillations in adjacent SMCs were no longer synchronized. BAPTA-AM, thapsigargin and U73122 hyperpolarized SMCs, relaxed the vessel, decreased basal calcium levels and abolished vasomotion. Chloride substitution abolished rhythmical activity, depolarized SMCs, increased basal calcium levels and constricted the vessel, while niflumic acid and DIDS abolished vasomotion. Ryanodine, charybdotoxin and TRAM-34, but not iberiotoxin, 4-aminopyridine or apamin, each depolarized SMCs and increased the frequency of rhythmical depolarizations and [Ca(2+)](i) oscillations. We conclude that vasomotion in the basilar artery depends on the release of intracellular calcium from IP(3) (inositol 1,4,5,-trisphosphate)-sensitive stores which activates calcium-dependent chloride channels to depolarize SMCs. Depolarization in turn activates voltage-dependent calcium channels, synchronizing contractions of adjacent cells through influx of extracellular calcium. Subsequent calcium-induced calcium release from ryanodine-sensitive stores activates an intermediate conductance potassium channel, hyperpolarizing the SMCs and providing a negative feedback pathway for regeneration of the contractile cycle.

Voltage-activated proton currents are reported for the first time in human peripheral blood T and B lymphocytes and in the human leukaemic T cell line Jurkat E6-1. The properties of H(+) currents studied using tight-seal voltage-clamp recording techniques were similar in all cells. Changing the pH gradient by one unit caused a 47 mV shift in the reversal potential, demonstrating high selectivity of the channels for protons. H(+) current activation upon membrane depolarisation had a sigmoidal time course that could be fitted by a single exponential function after a brief delay. Increasing pH(o) shifted the activation threshold to more negative potentials, and increased both the H(+) current amplitude and the rate of activation. In lymphocytes studied at pH(i) 6.0, the activation threshold was more negative and the H(+) current density was three times larger than at pH(i) 7.0. Increasing the intracellular Ca(2+) concentration to 1 microM did not change H(+) current amplitude or kinetics detectably. Extracellularly applied Zn(2+) and Cd(2+) inhibited proton currents, slowing activation and shifting the voltage-activation curve to more positive potentials. The H(+) current amplitude was 100 times larger in CD19+ B lymphocytes and in Jurkat E6-1 cells than in CD3+ T lymphocytes. Following stimulation with the phorbol ester PMA, the H(+) current density in peripheral blood T lymphocytes and Jurkat T cells increased. In contrast, the H(+) current density of phorbol ester (PMA)-stimulated B lymphocytes was reduced and activation became slower. The pattern of expression of H(+) channels in lymphocytes appears well suited to their proposed role of charge compensation during the respiratory burst.