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K IR2.1 potassium channels, producing inward rectifier potassium current (I K1), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal K IR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. K IR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within K IR2.1 sequences using the "epestfind" webtool. WT and ΔPEST K IR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. K IR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine K IR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the K IR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in K IR2.2, K IR2.3, and K IR2.6 proteins. Deletion of the PEST domain in California kingsnake and human K IR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T1/2 from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical I K1, however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of K IR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of I K1 channels.

KATP channels are metabolic sensors that couple cell energetics to membrane excitability. In pancreatic β-cells, channels formed by SUR1 and Kir6.2 regulate insulin secretion and are the targets of antidiabetic sulfonylureas. Here, we used cryo-EM to elucidate structural basis of channel assembly and gating. The structure, determined in the presence of ATP and the sulfonylurea glibenclamide, at ~6 Å resolution reveals a closed Kir6.2 tetrameric core with four peripheral SUR1s each anchored to a Kir6.2 by its N-terminal transmembrane domain (TMD0). Intricate interactions between TMD0, the loop following TMD0, and Kir6.2 near the proposed PIP2 binding site, and where ATP density is observed, suggest SUR1 may contribute to ATP and PIP2 binding to enhance Kir6.2 sensitivity to both. The SUR1-ABC core is found in an unusual inward-facing conformation whereby the two nucleotide binding domains are misaligned along a two-fold symmetry axis, revealing a possible mechanism by which glibenclamide inhibits channel activity. DOI: http://dx.doi.org/10.7554/eLife.24149.001, eLife digest The hormone insulin reduces blood sugar levels by encouraging fat, muscle and other body cells to take up sugar. When blood sugar levels rise following a meal, cells within the pancreas known as beta cells should release insulin. In people with diabetes, the beta cells fail to release insulin, meaning that the high blood sugar levels are not corrected. When blood sugar levels are high, beta cells generate more energy in the form of ATP molecules. The increased level of ATP causes channels called ATP-sensitive potassium (KATP) channels in the membrane of the cell to close. This triggers a cascade of events that leads to the release of insulin. Some treatments for diabetes alter how the KATP channels work. For example, a widely prescribed medication called glibenclamide (also known as glyburide in the United States) stimulates the release of insulin by preventing the flow of potassium through KATP channels. It remains unknown exactly how ATP and glibenclamide interact with the channel’s molecular structure to stop the flow of potassium ions. KATP channels are made up of two proteins called SUR1 and Kir6.2. To investigate the structure of the KATP channel, Martin et al. purified channels made of the hamster form of the SUR1 protein and the mouse form of Kir6.2, which each closely resemble their human counterparts. The channels were purified in the presence of ATP and glibenclamide and were then rapidly frozen to preserve their structure, which allowed them to be visualized individually using electron microscopy. By analyzing the images taken from many channels, Martin et al. constructed a highly detailed, three-dimensional map of the KATP channel. The structure revealed by this map shows how SUR1 and Kir6.2 work together and provides insight into how ATP and glibenclamide interact with the channel to block the flow of potassium, and hence stimulate the release of insulin. An important next step will be to improve the structure to more clearly identify where ATP and glibenclamide bind to the KATP channel. It will also be important to study the structures of channels that are bound to other regulatory molecules. This will help researchers to fully understand how KATP channels located throughout the body operate under healthy and diseased conditions. This knowledge will aid in the design of more effective drugs to treat several devastating diseases caused by defective KATP channels. DOI: http://dx.doi.org/10.7554/eLife.24149.002

The hormone insulin reduces blood sugar levels by encouraging fat, muscle and other body cells to take up sugar. When blood sugar levels rise following a meal, cells within the pancreas known as beta cells should release insulin. In people with diabetes, the beta cells fail to release insulin, meaning that the high blood sugar levels are not corrected. When blood sugar levels are high, beta cells generate more energy in the form of ATP molecules. The increased level of ATP causes channels called ATP-sensitive potassium (KATP) channels in the membrane of the cell to close. This triggers a cascade of events that leads to the release of insulin. Some treatments for diabetes alter how the KATP channels work. For example, a widely prescribed medication called glibenclamide (also known as glyburide in the United States) stimulates the release of insulin by preventing the flow of potassium through KATP channels. It remains unknown exactly how ATP and glibenclamide interact with the channel’s molecular structure to stop the flow of potassium ions. KATP channels are made up of two proteins called SUR1 and Kir6.2. To investigate the structure of the KATP channel, Martin et al. purified channels made of the hamster form of the SUR1 protein and the mouse form of Kir6.2, which each closely resemble their human counterparts. The channels were purified in the presence of ATP and glibenclamide and were then rapidly frozen to preserve their structure, which allowed them to be visualized individually using electron microscopy. By analyzing the images taken from many channels, Martin et al. constructed a highly detailed, three-dimensional map of the KATP channel. The structure revealed by this map shows how SUR1 and Kir6.2 work together and provides insight into how ATP and glibenclamide interact with the channel to block the flow of potassium, and hence stimulate the release of insulin. An important next step will be to improve the structure to more clearly identify where ATP and glibenclamide bind to the KATP channel. It will also be important to study the structures of channels that are bound to other regulatory molecules. This will help researchers to fully understand how KATP channels located throughout the body operate under healthy and diseased conditions. This knowledge will aid in the design of more effective drugs to treat several devastating diseases caused by defective KATP channels.

The development of integral membrane protein cell-free synthesis permits in-vitro labeling of accessible cysteines for real-time FRET and LRET measurements. The functional integrity of these synthetic ion channel proteins has been verified at the whole oocyte level by direct injection into, and recording from, Xenopus oocytes. However, the microscopic single-channel properties of cell-free translated protein have not been systematically examined. In the present study, we compare patch-clamp currents originating from cell-free protein with currents derived from mRNA injection, using the same (single-Cys) inward rectifier DNA template (C189-Kir1.1b). Results indicate that cell-free Kir protein, incorporated into liposomes and injected into oocytes, is trafficked to the plasma membrane where it inserts in an outside-out orientation and exhibits single-channel characteristics identical to that derived from a corresponding mRNA.

Inwardly rectifying K+ channels (Kir) comprise seven subfamilies that can be subdivided further on the basis of cytosolic pH (pHi) sensitivity, rectification strength and kinetics, and resistance to run-down. Although distinct residues within each channel subunit define these properties, heteromeric association with other Kir subunits can modulate them. We identified such an effect in the wild-type forms of Kir4.2 and Kir5.1 and used this to further understand how the functional properties of Kir channels relate to their structures. Kir4.2 and a Kir4.2–Kir5.1 fusion protein were expressed in HEK293 cells. Inward currents from Kir4.2 were stable over 10 min and pHi-insensitive (pH 6 to 8). Conversely, currents from Kir4.2–Kir5.1 exhibited a pHi-sensitive run-down at slightly acidic pHi. At pHi 7.2, currents in response to voltage steps positive to EK were essentially time independent for Kir4.2 indicating rapid block by Mg2+. Coexpression with Kir5.1 significantly increased the blocking time constant, and increased steady-state outward current characteristic of weak rectifiers. Recovery from blockade at negative potentials was voltage dependent and 2 to 10 times slower in the homomeric channel. These results show that Kir5.1 converts Kir4.2 from a strong to a weak rectifier, rendering it sensitive to pHi, and suggesting that Kir5.1 plays a role in fine-tuning Kir4.2 activity.

Small organic amines block open voltage-gated K+ channels and can be trapped by subsequent closure. Such studies provide strong evidence for voltage gating occurring at the intracellular end of the channel. We engineered the necessary properties (long block times with unblock kinetics comparable to, or slower than, the kinetics of gating) into spermine-blocked, ATP-gated (N160D,L157C) mutant KATP channels, in order to test the possibility of “blocker trapping” in ligand-gated Kir channels. Spermine block of these channels is very strongly voltage dependent, such that, at positive voltages, the off-rate of spermine is very low. A brief pulse to negative voltages rapidly relieves the block, but no such relief is observed in ATP-closed channels. The results are well fit by a simple kinetic model that assumes no spermine exit from closed channels. The results incontrovertibly demonstrate that spermine is trapped in channels that are closed by ATP, and implicate the M2 helix bundle crossing, or somewhere lower, as the probable location of the gate.

Arginine residue at position 285 (R285) in the intracellular C-terminal domain of inward rectifier potassium channel Kir2.2 is conserved in many species, but missing in previously reported human Kir2.2 sequences. We here identified the human Kir2.2 gene in normal individuals, which contained R285 in the deduced amino-acid sequence (hKir2.2/R285). All 30 individuals we examined were homozygous for Kir2.2/R285 gene. The hKir2.2/R285 was electrophysiologically functional in both mammalian cells and Xenopus oocytes. However, the hKir2.2 missing R285 was functional only in Xenopus oocytes, but not in mammalian cells. Thus, R285 in Kir2.2 is important for its functional expression in mammalian cells.

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. KIR 2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (IK1 ), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late-endosome/lysosome system. Here we defined the potential interference in KIR 2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited IK1 in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK-KWGF cells, both drugs dose- and time-dependently increased KIR 2.1 expression (2.0 ± 0.2-fold with amiodarone: 10 μM, 24 hrs; 2.3 ± 0.3-fold with dronedarone: 5 μM, 24 hrs) and late-endosomal/lysosomal KIR 2.1 accumulation. Increased KIR 2.1 expression level was also observed in the presence of Nav 1.5 co-expression. Augmented KIR 2.1 protein levels and intracellular accumulation were also observed in COS-7, END-2, MES-1 and EPI-7 cells. Both drugs had no effect on Kv 11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at -120 mV, 5 μM) enhanced IKIR2.1 upon 24-hrs treatment, whereas dronedarone tended to increase IKIR2.1 and it did not reach significance (43.8 ± 5.5%, P = 0.26 at -120 mV; 2 μM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced IK1 by inhibiting KIR 2.1 degradation.

Inwardly-rectifying potassium (Kir) channels contribute to maintenance of the resting membrane potential and regulation of electrical excitation in many cell types. Strongly rectifying Kir channels exhibit a very steep voltage dependence resulting in silencing of their activity at depolarized membrane voltages. The mechanism underlying this steep voltage dependence is blockade by endogenous polyamines. These small multifunctional, polyvalent metabolites enter the long Kir channel pore from the intracellular side, displacing multiple occupant ions as they migrate to a stable binding site in the transmembrane region of the channel. Numerous structure-function studies have revealed structural elements of Kir channels that determine their susceptibility to polyamine block, and enable the steep voltage dependence of this process. In addition, various channelopathies have been described that result from alteration of the polyamine sensitivity or activity of strongly rectifying channels. The primary focus of this article is to summarize current knowledge of the molecular mechanisms of polyamine block, and provide some perspective on lingering uncertainties related to this physiologically important mechanism of ion channel blockade. We also briefly review some of the important and well understood physiological roles of polyamine sensitive, strongly rectifying Kir channels, primarily of the Kir2 family.

Use of randomized peptide libraries to evolve molecules with new functions provides a means for developing novel regulators of protein activity. Despite the demonstrated power of such approaches for soluble targets, application of this strategy to membrane systems, such as ion channels, remains challenging. Here, we have combined libraries of a tethered protein scaffold with functional selection in yeast to develop a novel activator of the G-protein-coupled mammalian inwardly rectifying potassium channel Kir3.2 (GIRK2). We show that the novel regulator, denoted N5, increases Kir3.2 (GIRK2) basal activity by inhibiting clearance of the channel from the cellular surface rather than affecting the core biophysical properties of the channel. These studies establish the tethered protein display strategy as a means to create new channel modulators and highlight the power of approaches that couple randomized libraries with direct selections for functional effects. Our results further underscore the possibility for the development of modulators that influence channel function by altering cell surface expression densities rather than by direct action on channel biophysical parameters. The use of tethered library selection strategies coupled with functional selection bypasses the need for a purified target and is likely to be applicable to a range of membrane protein systems.

Ionic currents related to the major potassium uptake systems in Saccharomyces cerevisiae were examined by whole cell patch-clamping, under K+ replete conditions. Those currents have the following properties. They (1) are inward under all conditions investigated, (2) arise instantaneously with appropriate voltage steps, (3) depend solely upon the moderate affinity transporter Trk2p, not upon the high affinity transporter Trk1p. They (4) appear to be independent of the extracellular K+ concentration, (5) are also independent of extracellular Ca2+, Mg2+ and Cl− but (6) are strongly dependent on extracellular pH, being large at low pH (up to several hundred pA at −200 mV and pH 4) and near zero at high pH (above 7.5). They (7) increase in proportion to log[H+]o, rather than directly in proportion to the proton concentration and (8) behave kinetically as if each transporter cycle moved one proton plus one (high pH) or two (low pH) other ions, as yet unidentified. In view of background knowledge on K+ transport related to Trk2p, the new results suggest that the K+ status of yeast cells modulates both the kinetics of Trk2p-mediated transport and the identity of ions involved. That modulation could act either on the Trk2 protein itself or on interactions of Trk2 with other proteins in a hypothetical transporter complex. Structural considerations suggest a strong analogy to the KtrAB system in Vibrio alginolyticus and/or the TrkH system in Escherichia coli.

Using cysteine (Cys) scanning mutagenesis of the inward rectifier K+ channel Kir2.1, we investigated its pore structure and putative conformational changes. In the background of the Kir2.1 mutant C149F which showed no sensitivity towards Cys-modifying reagents, Cys residues were introduced at 10 positions in the H5 pore region. Out of six functional mutants, T141C and F147C showed clear changes in current amplitude when Cys-modifying reagents were applied from the external side. These results suggest that the corresponding sections of the H5 pore region face to the external side which is in contrast to the results previously obtained for voltage-gated K+ (Kv) channels. Using the mutants T141C and F147C, we investigated whether or not Kir2.1 channels show state-dependent conformational changes of the pore structure. Substantial alterations of the holding potential or external K+ concentration, however, did not cause any significant change in the speed of channel modification upon application of Cys-specific reagents, suggesting that Kir2.1 channels do not undergo conformational changes, in contrast to C-type inactivating Kv channels.

Inward rectification induced by mono- and diaminoalkane application to inside-out membrane patches was studied in Kir2.1 (IRK1) channels expressed in Xenopus oocytes. Both monoamines and diamines block Kir2.1 channels, with potency increasing as the alkyl chain length increases (from 2 to 12 methylene groups), indicating a strong hydrophobic interaction with the blocking site. For diamines, but not monoamines, increasing the alkyl chain also increases the steepness of the voltage dependence, at any concentration, from a limiting minimal value of approximately 1.5 (n = 2 methylene groups) to approximately 4 (n = 10 methylene groups). These observations lead us to hypothesize that monoamines and diamines block inward rectifier K+ channels by entering deeply into a long, narrow pore, displacing K+ ions to the outside of the membrane, with this displacement of K+ ions contributing to "extra" charge movement. All monoamines are proposed to lie with the "head" amine at a fixed position in the pore, determined by electrostatic interaction, so that zdelta is independent of monoamine alkyl chain length. The head amine of diamines is proposed to lie progressively further into the pore as alkyl chain length increases, thus displacing more K+ ions to the outside, resulting in charge movement (zdelta) increasing with the increase in alkyl chain length.

A voltage-gated K+ conductance resembling that of the human ether-à-go-go-related gene product (HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K+ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K+ conductance-voltage relation was half maximal at -59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive (-14 vs. -39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:795-805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs+ or Ba2+ occurred only at millimolar concentrations, La3+ blocked with Ki = approximately 40 microM, and the HERG-selective blocker, E-4031, blocked with Ki = 37 nM. Implications of the presence of HERG-like K+ channels for the ontogeny of microglia are discussed.

In order to further investigate a G-protein activated inwardly rectifying potassium channel subunit, GIRK1 was expressed in Xenopus oocytes (where it coassembles with the endogenous GIRK5). The mechanism underlying ion permeation and rectification were measured in isolated inside-out patches. Single channel current amplitudes under symmetrical K+ concentrations at different holding potentials were evaluated. Inward-rectification of K+-currents through open GIRK1/GIRK5 channels was removed by washing out polyamines and Mg2+ ions. We developed a simple `two-sites-three-barrier' (2S3B) Eyring rate theory model of K+ ion permeation for GIRK1/GIRK5 channels. The resulting optimized parameter-set will be used as a working model for subsequent investigation regarding K+ permeation process through the GIRK1/GIRK5 channel.

ATP-sensitive potassium (KATP) channels link cellular metabolism to electrical activity in nerve, muscle, and endocrine tissues. They are formed as a functional complex of two unrelated subunits—a member of the Kir inward rectifier potassium channel family, and a sulfonylurea receptor (SUR), a member of the ATP-binding cassette transporter family, which includes cystic fibrosis transmembrane conductance regulators and multidrug resistance protein, regulators of chloride channel activity. This recent discovery has brought together proteins from two very distinct superfamilies in a novel functional complex. The pancreatic KATP channel is probably formed specifically of Kir6.2 and SUR1 isoforms. The relationship between SUR1 and Kir6.2 must be determined to understand how SUR1 and Kir6.2 interact to form this unique channel. We have used mutant Kir6.2 subunits and dimeric (SUR1-Kir6.2) constructs to examine the functional stoichiometry of the KATP channel. The data indicate that the KATP channel pore is lined by four Kir6.2 subunits, and that each Kir6.2 subunit requires one SUR1 subunit to generate a functional channel in an octameric or tetradimeric structure.

We have investigated the subunit stoichiometry of the pancreatic beta-cell ATP-sensitive K+ (KATP) channel (SUR1/Kir6.2 channel) by constructing cDNA encoding a single polypeptide (beta alpha polypeptide) consisting of a SUR1 (beta) subunit and a Kir6.2 (alpha) subunit. 86Rb+ efflux and single-channel properties of COS1 cells expressing beta alpha polypeptides were similar to those of COS1 cells coexpressing alpha monomers and beta monomers. Coexpression of beta alpha polypeptides with alpha monomers inhibited the K+ currents, while coexpression with beta monomers did not. We then constructed another single polypeptide (beta alpha2) consisting of a beta subunit and a dimeric repeat of the alpha subunit. 86Rb+ efflux from COS1 cells expressing beta alpha2 polypeptides was small, but was restored by supplementation with beta monomers. These results indicate that the activity of K(ATP) channels is optimized when the alpha and beta subunits are coexpressed with a molar ratio of 1:1. Since inward rectifier K+ channels are thought to function as homo- or hetero-tetramers, this suggests that the K(ATP) channel functions as a multimeric protein, most likely a hetero-octamer composed of a tetramer of the Kir6.2 subunit and a tetramer of the SUR1 subunit.

In this study we examined long-lasting effects mediated by intracellular mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) on two voltage dependent potassium conductances in CA1 pyramidal neurons, i.e. the transient current I(A) and the delayed rectifier, and on the inward rectifier I(Q), a mixed sodium/potassium current. All experiments were carried out in hippocampal slices with the in situ patch clamp technique, in the whole cell mode. Neurons recorded 30 min to 3 h after a brief application of 30 nM corticosterone to slices from adrenalectomized rats, thus saturating MRs and occupying most of the GRs, displayed a large I(Q)-conductance similar to neurons in slices from the sham-operated controls. By contrast, if only MRs or only GRs were activated, the I(Q)-conductance was significantly smaller than for the corticosterone-treated group of cells, indicating that simultaneous activation of both MRs and GRs is necessary to achieve a large I(Q)-conductance. If corticosterone was applied in the presence of a protein synthesis inhibitor, the I(Q) conductance was significantly smaller than in the absence of the inhibitor. Properties of the I(A) and the delayed rectifier were not affected by the various corticosteroid treatments. In conclusion, the data indicate that in particular the I(Q)-conductance is under a gene-mediated control of corticosteroid hormones. The I(Q)-conductance is relatively low when only MRs are activated, as occurs for the rat in the morning under rest, and high when both MRs and GRs are occupied, as occurs at the peak of the circadian cycle and after stress. This finding suggests that MR- and GR-mediated events act in synergism to control the I(Q), thus contributing to regulation of cellular excitability under physiologically relevant conditions.

The family of voltage-gated (Shaker-like) potassium channels in plants includes both inward-rectifying (Kin) channels that allow plant cells to accumulate K+ and outward-rectifying (Kout) channels that mediate K+ efflux. Despite their close structural similarities, Kin and Kout channels differ in their gating sensitivity towards voltage and the extracellular K+ concentration. We have carried out a systematic program of domain swapping between the Kout channel SKOR and the Kin channel KAT1 to examine the impacts on gating of the pore regions, the S4, S5, and the S6 helices. We found that, in particular, the N-terminal part of the S5 played a critical role in KAT1 and SKOR gating. Our findings were supported by molecular dynamics of KAT1 and SKOR homology models. In silico analysis revealed that during channel opening and closing, displacement of certain residues, especially in the S5 and S6 segments, is more pronounced in KAT1 than in SKOR. From our analysis of the S4–S6 region, we conclude that gating (and K+-sensing in SKOR) depend on a number of structural elements that are dispersed over this ∼145-residue sequence and that these place additional constraints on configurational rearrangement of the channels during gating. Fil: Riedelsberger, Janin . Universität Potsdam; Alemania. Max-Planck Institute of Molecular Plant Physiology; Alemania Fil: Sharma, Tripti . Universität Potsdam; Alemania. Max-Planck Institute of Molecular Plant Physiology; Alemania Fil: Gonzalez, Wendy . Universidad de Talca; Chile Fil: Gajdanowicz, Pawel . Universität Potsdam; Alemania Fil: Morales Navarro, Samuel Elías . Universidad de Talca; Chile Fil: Garcia-Mata, Carlos. University Of Glasgow; Reino Unido. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mueller Roeber, Bernd. Max-Planck Institute of Molecular Plant Physiology; Alemania. Universität Potsdam; Alemania Fil: González Nilo, Fernando Danilo . Universidad de Talca; Chile Fil: Blatt, Michael R. . University Of Glasgow; Reino Unido Fil: Dreyer, Ingo . Universität Potsdam; Alemania

This work demonstrates that extracellular Na(+) modulates the cloned inwardly rectifying K(+) channels Kir4.1 and Kir4.1-Kir5.1. Whole-cell patch clamp studies on astrocytes have previously indicated that inward potassium currents are regulated by external Na(+). We expressed Kir4.1 and Kir4.1-Kir5.1 in Xenopus oocytes to disclose if Kir4.1 and/or Kir4.1-Kir5.1 at the molecular level are responsible for the observed effect of [Na(+)](o) and to investigate the regulatory mechanism of external cations further. Our results showed that Na(+) has a biphasic modulatory effect on both Kir4.1 and Kir4.1-Kir5.1 currents. Depending on the Na(+)-concentration and applied voltage, the inward Kir4.1/Kir4.1-Kir5.1 currents are either enhanced or reduced by extracellular Na(+). The Na(+) activation was voltage-independent, whereas the Na(+)-induced reduction of the Kir4.1 and Kir4.1-Kir5.1 currents was both concentration-, time- and voltage-dependent. Our data indicate that the biphasic effect of extracellular Na(+)on the Kir4.1 and Kir4.1-Kir5.1 channels is caused by two separate mechanisms.

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K+ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K+ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K+ equilibrium potential (EK), (2)decreased in slope conductance near EK, (3) was dependent on [K+]o and (4) was blocked by external Ba2+ and Cs+. These properties were similar to those of the inwardly rectifying K+ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K+ channels.

gamma-Aminobutyric acid (GABA)(B) receptors are known to enhance activation of Kir3 channels generating G-protein-dependent inward rectifier K(+)-currents (GIRK). In some neurons, GABA(B) receptors either cause a tonic GIRK activation or generate a late K(+)-dependent inhibitory postsynaptic current component. However, other neurons express Kir2 channels, which generate a constitutive inward rectifier K(+)-current (CIRK) without requiring G-protein activation. The functional coupling of CIRK with GABA(B) receptors remained unexplored so far. About 50% of rat cerebellar granule cells in the internal granular layer of P19-26 rats showed a sizeable CIRK current. Here, we have investigated CIRK current regulation by GABA(B) receptors in cerebellar granule cells, which undergo GABAergic inhibition through Golgi cells. By using patch-clamp recording techniques and single-cell reverse transcriptase-polymerase chain reaction in acute cerebellar slices, we show that granule cells co-express Kir2 channels and GABA(B) receptors. CIRK current biophysical properties were compatible with Kir2 but not Kir3 channels, and could be inhibited by the GABA(B) receptor agonist baclofen. The action of baclofen was prevented by the GABA(B) receptor blocker CGP35348, involved a pertussis toxin-insensitive G-protein-mediated pathway, and required protein phosphatases inhibited by okadaic acid. GABA(B) receptor-dependent CIRK current inhibition could also be induced by repetitive GABAergic transmission at frequencies higher than the basal autorhythmic discharge of Golgi cells. These results suggest therefore that GABA(B) receptors can exert an inhibitory control over CIRK currents mediated by Kir2 channels. CIRK inhibition was associated with an increased input resistance around rest and caused a approximately 5 mV membrane depolarization. The pro-excitatory action of these effects at an inhibitory synapse may have an homeostatic role re-establishing granule cell readiness under conditions of strong inhibition., Journal Article, Research Support, Non-U.S. Gov't, FLWIN, info:eu-repo/semantics/published

Animal and plant voltage-gated ion channels share a common architecture. They are made up of four subunits and the positive charges on helical S4 segments of the protein in animal K+ channels are the main voltage-sensing elements. The KAT1 channel cloned from Arabidopsis thaliana, despite its structural similarity to animal outward rectifier K+ channels is, however, an inward rectifier. Here we detected KAT1-gating currents due to the existence of an intrinsic voltage sensor in this channel. The measured gating currents evoked in response to hyperpolarizing voltage steps consist of a very fast (tau = 318 +/- 34 micros at -180 mV) and a slower component (4.5 +/- 0.5 ms at -180 mV) representing charge moved when most channels are closed. The observed gating currents precede in time the ionic currents and they are measurable at voltages (less than or equal to -60) at which the channel open probability is negligible ( approximately 10-4). These two observations, together with the fact that there is a delay in the onset of the ionic currents, indicate that gating charge transits between several closed states before the KAT1 channel opens. To gain insight into the molecular mechanisms that give rise to the gating currents and lead to channel opening, we probed external accessibility of S4 domain residues to methanethiosulfonate-ethyltrimethylammonium (MTSET) in both closed and open cysteine-substituted KAT1 channels. The results demonstrate that the putative voltage-sensing charges of S4 move inward when the KAT1 channels open.

We distinguished four distinct groups of megakaryocytic cells on the basis of their voltage-gated membrane currents. One group of 32 cells (15%), exhibited an inward rectifying current and had a diameter of 12 +/- 3.5 microm (mean +/- S.D.). A large group of 85 cells (39%) exhibited only a 'leakage-like' current and had a diameter of 15.8 +/- 3.7 microm. The other two groups of cells exhibited voltage-gated outward currents. One group consisted of 43 'I-type' cells (19%), with a diameter of 22.3 +/- 3.4 microm, for which the maximal outward current occurred for a voltage step from -60 to either 0 or +20 mV. For the last group of 60 'M-type' cells (27%), which had a diameter of 26.7 +/- 2.9 microm, the maximal outward current occurred for a voltage step from -60 to +80 mV, the largest voltage step used. The currents recorded in this study, from megakaryocytes having 'leakage-like' currents and 'I-type' currents, were indistinguishable from the voltage-gated currents of the megakaryocytes from myelogenous leukemia patients, in which voltage-gated currents were suppressed (Kapural, L., O'Rourke, F., Feinstein, M.B. and Fein, A. (1995) Blood 86, 1043), suggesting that the megakaryocytes from the myelogenous leukemia patients are a dedifferentiated or less mature form of megakaryocyte.

ATP-sensitive K+ channels comprise a complex of at least two proteins: a member of the inwardly rectifying Kir6 family (e.g. Kir6.2) and a sulphonylurea receptor (e.g. SUR1) which belongs to the ATP-binding cassette (ABC) superfamily. Using specific radiolabeled antisense oligonucleotides, the cellular localization of both mRNAs was investigated in the rodent brain by in situ hybridization. The distribution of both transcripts was widespread throughout the brain and showed a high degree of overlap with peak expression levels in the hippocampus, neocortex, olfactory bulb, cerebellum, and several distinct nuclei of the midbrain and brainstem, indicating their important role in vital brain function.

Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential record were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two slates. The majority of the guard cells had depolarized membrane potentials. which were largely dependent on external K+ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (E-m) was not sensitive to the external K+ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K+-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235-246). The activation and inactivation times and the activation potential, all depended on the reversal potential (E-rev) of the s-ORC conductance. The s-ORC was blocked by Ba2+ (K-1/2 = 0.3-1.3 mM) and verapamil (K-1/2 = 15-20 mu M). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba2+. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized stale. The activation time and activation potential of this channel were not sensitive to the external K+ concentration. The slow activation of the IRC (t(1/2) approximate to 0.5 s) and its negative activation potential (V-threshold = -155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl ct al., 1995, Free Natl Acad Sci USA 92: 2701-2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes.

Large subtype-specific differences in the sensitivity of cloned inward-rectifier K+ channels of the IRK1, BIR10 and ROMK1 subtype to being blocked by intracellular spermine (SPM) are described. It is shown, by site-directed mutagenesis, that the four orders of magnitude larger SPM sensitivity of BIR10 channels compared to ROMK1 channels may be explained by a difference in a single amino acid in the putative transmembrane segment TMII. This residue, a negatively charged glutamate in BIR10, is homologous to the residue in IRK1 and ROMK1 which has previously been shown to change gating properties and Mg2+ sensitivity. Differential block by physiological SPM concentrations is suggested as a major functional difference between subtypes of inward-rectifier K+ channels.

We have isolated a cDNA coding for an inward rectifier K+ channel (RBHIK1) from rabbit heart. The cloned cDNA encodes a protein of 427 amino acids with two putative transmembrane segments. The primary structure of RBHIK1 is highly homologous to that of IRK1 which is an inward rectifier K+ channel recently cloned from mouse macrophage by expression cloning. When expressed in Xenopus oocytes, RBHIK1 current showed strong inward rectification and was inhibited by extracellular Ba2+ and Cs+. RNA blot analysis revealed the expression of RBHIKl mRNA in various rabbit tissues, especially high level in the ventricular muscle.

The mouse potassium channel Kir2.3 possesses conserved extracellular cysteine residues at positions 113 and 145. We have investigated the role of these cysteines in structure/function and membrane trafficking. Cysteine to serine mutations resulted in the absence of potassium currents in oocytes and co-expression of these mutants with wild-type channel showed a dominant negative inhibition of wild-type currents. FLAG-tagged channels expressed in oocytes were detected in the cell membrane by anti-FLAG antibody for wild-type and mutant channels. In vitro translation using the reticulocyte lysate system showed that mutation of these residues did not affect processing nor insertion into membranes. Cysteine residues at 113 and 145 are therefore required for function of the Kir2.3 channel but not for processing into the cell membrane; disulfide bonds between subunits are unlikely.

The plasma membrane of the yeast Saccharomyces cerevisiae possesses a non-specific cation 'channel', tentatively dubbed NSC1, which is blocked by normal (mM) calcium and other divalent metal ions, is unblocked by reduction of extracellular free divalents below approximately 10 microM, and is independent of the identified potassium channel and porters in yeast, Duk1p, Trk1p, and Trk2p. Ion currents through NSC1, observed by means of whole-cell patch recording, have the following characteristics: Large amplitude, often exceeding 1 nA of K+/ cell at -200 mV, in tetraploid yeast, sufficient to double the normal intracellular K+ concentration within 10 s; non-saturation at large negative voltages; complicated activation kinetics, in which approximately 50% of the total current arises nearly instantaneously with a voltage-clamp step, while the remainder develops as two components, with time constants of approximately 100 ms and approximately 1.3 s; and voltage independence of both the activation time constants and the associated fractional current amplitudes.

Locus control regions (LCRs) are cis-acting elements that confer position-independent and copy-number-dependent expression upon associated genes in transgenic mice. Here we show the second example of the use of an LCR (the rat LAP LCR) in a stable expression vector system, used here in conjunction with the rat liver (NRLM) cell line. Non-transfected NRLM cells are electrically silent and highly suitable for patch clamp electrophysiology. We report reliable constitutive expression from two different K+ channel cDNAs; the voltage-gated rat clone Kv3.4 and the inward rectifier mouse clone Kir2.1. We further show that constitutive expression levels are stable for at least 8 weeks from initial recording.

CO2 chemosensing is a vital function for the\ud maintenance of life that helps to control acid–base balance.\ud Most studies have reported that CO2 is measured via its\ud proxy, pH. Here we report an inwardly rectifying channel,\ud in outside-out excised patches from HeLa cells that was\ud sensitive to modest changes in PCO2 under conditions of\ud constant extracellular pH. As PCO2 increased, the open\ud probability of the channel increased. The single-channel\ud currents had a conductance of 6.7 pS and a reversal\ud potential of –70 mV, which lay between the K+ and Cl–\ud equilibrium potentials. This reversal potential was shifted\ud by +61 mV following a tenfold increase in extracellular\ud [K+] but was insensitive to variations of extracellular [Cl–].\ud The single-channel conductance increased with extracellular\ud [K+]. We propose that this channel is a member of the\ud Kir family. In addition to this K+ channel, we found that\ud many of the excised patches also contained a conductance\ud carried via a Cl–-selective channel. This CO2-sensitive Kir\ud channel may hyperpolarize excitable cells and provides a\ud potential mechanism for CO2-dependent inhibition during\ud hypercapnia.

Highlights ► ACh and ET-1 activate a K+ current in cardiac atrioventricular nodal cells. ► Tertiapin-Q sensitive IKACh activated via M2 receptors shows bi-exponential ‘fade’. ► ET-1 activates a similar current that also fades. ► The fade reflects desensitization rather than altered K+ ion driving force. ► Acetylcholine is able to cross-desensitize the AVN cell response to endothelin-1., The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, IKACh. However, it is not yet known if this response exhibits ‘fade’ or desensitization in the AVN, as established for the heart’s primary pacemaker – the sinoatrial node. In this study, acute activation of IKACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37 °C. 0.1–1 μM acetylcholine (ACh) rapidly activated a robust IKACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300 nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1 μM). During sustained ACh exposure the elicited IKACh exhibited bi-exponential fade (τf of 2.0 s and τs 76.9 s at −120 mV; 1 μM ACh). 10 nM ET-1 elicited a current similar to IKACh, which faded with a mono-exponential time-course (τ of 52.6 s at −120 mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization.