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Unipolar brush cells (UBCs) are excitatory granular layer interneurons in the vestibulocerebellum. Here we assessed motor coordination and balance to investigate if deletion of acid-sensing ion channel 5 (Asic5), which is richly expressed in type II UBCs, is sufficient to cause ataxia. The possible cellular mechanism underpinning ataxia in this global Asic5 knockout model was elaborated using brain slice electrophysiology. Asic5 deletion impaired motor performance and decreased intrinsic UBC excitability, reducing spontaneous action potential firing by slowing maximum depolarization rate. Reduced intrinsic excitability in UBCs was partially compensated by suppression of the magnitude and duration of delayed hyperpolarizing K+ currents triggered by glutamate. Glutamate typically stimulates burst firing subsequent to this hyperpolarization in normal type II UBCs. Burst firing frequency was elevated in knockout type II UBCs because it was initiated from a more depolarized potential compared to normal cells. Findings indicate that Asic5 is important for type II UBC activity and that loss of Asic5 contributes to impaired movement, likely, at least in part, due to altered temporal processing of vestibular input.

Because of its cardio-protective effects, a low Na, high K diet (LNaHK) is often warranted in conjunction with diuretics to treat hypertensive patients. However, it is necessary to understand the renal handling of such diets in order to choose the best diuretic. Wild type (WT) or Renal Outer Medullary K channel (ROMK) knockout mice (KO) were given a regular (CTRL), LNaHK, or high K diet (HK) for 4–7 days. On LNaHK, mice treated with either IP furosemide for 12 hrs, or given furosemide in drinking water for 7 days, exhibited decreased K clearance. We used free-flow micropuncture to measure the [K+] in the early distal tubule (EDT [K+]) before and after furosemide treatment. Furosemide increased the EDT [K+] in WT on CTRL but decreased that in WT on LNaHK. Furosemide did not affect the EDT [K+] of KO on LNaHK or WT on HK. Furosemide-sensitive Na+ excretion was significantly greater in mice on LNaHK than those on CTRL or HK. Patch clamp analysis of split-open TALs revealed that 70-pS ROMK exhibited a higher open probability (Po) but similar density in mice on LNaHK, compared with CTRL. No difference was found in the density or Po of the 30 pS K channels between two groups. These results indicate mice on LNaHK exhibited furosemide-sensitive net K+ secretion in the TAL that is dependent on increased NKCC2 activity and mediated by ROMK. We conclude that furosemide is a K-sparing diuretic by decreasing the TAL net K+ secretion in subjects on LNaHK.

Chemotherapy induced peripheral neuropathy (CIPN), a side effect of many anti-cancer drugs including the vinca alkaloids, is characterized by a severe pain syndrome that compromises treatment in many patients. Currently there are no effective treatments for this pain syndrome except for the reduction of anti-cancer drug dose. Existing data supports the model that the pain associated with CIPN is the result of anti-cancer drugs augmenting the function of the peripheral sensory nociceptors but the cellular mechanisms underlying the effects of anti-cancer drugs on sensory neuron function are not well described. Studies from animal models have suggested a number of disease etiologies including mitotoxicity, axonal degeneration, immune signaling, and reduced sensory innervations but these outcomes are the result of prolonged treatment paradigms and do not necessarily represent the early formative events associated with CIPN. Here we show that acute exposure to vinca alkaloids results in an immediate pain syndrome in both flies and mice. Furthermore, we demonstrate that exposure of isolated sensory neurons to vinca alkaloids results in the generation of an inward sodium current capable of depolarizing these neurons to threshold resulting in neuronal firing. These neuronal effects of vinca alkaloids require the transient receptor potential ankyrin-1 (TrpA1) channel, and the hypersensitization to painful stimuli in response to the acute exposure to vinca alkaloids is reduced in TrpA1 mutant flies and mice. These findings demonstrate the direct excitation of sensory neurons by CIPN-causing chemotherapy drugs, and identify TrpA1 as an important target during the pathogenesis of CIPN.

The voltage-gated K(+) (Kv) channel blocker 4-aminopyridine (4-AP) is used to target symptoms of the neuroinflammatory disease multiple sclerosis (MS). By blocking Kv channels, 4-AP facilitates action potential conduction and neurotransmitter release in presynaptic neurons, lessening the effects of demyelination. Because they conduct inward Na(+) and Ca(2+) currents that contribute to axonal degeneration in response to inflammatory conditions, acid-sensing ion channels (ASICs) contribute to the pathology of MS. Consequently, ASICs are emerging as disease-modifying targets in MS. Surprisingly, as first demonstrated here, 4-AP inhibits neuronal degenerin/epithelial Na(+) (Deg/ENaC) channels, including ASIC and BLINaC. This effect is specific for 4-AP compared with its heterocyclic base, pyridine, and the related derivative, 4-methylpyridine; and akin to the actions of 4-AP on the structurally unrelated Kv channels, dose- and voltage-dependent. 4-AP has differential actions on distinct ASICs, strongly inhibiting ASIC1a channels expressed in central neurons but being without effect on ASIC3, which is enriched in peripheral sensory neurons. The voltage dependence of the 4-AP block and the single binding site for this inhibitor are consistent with 4-AP binding in the pore of Deg/ENaC channels as it does Kv channels, suggesting a similar mechanism of inhibition in these two classes of channels. These findings argue that effects on both Kv and Deg/ENaC channels should be considered when evaluating the actions of 4-AP. Importantly, the current results are consistent with 4-AP influencing the symptoms of MS as well as the course of the disease because of inhibitory actions on Kv and ASIC channels, respectively.

The molecular transformation of an external stimulus into changes in sensory neuron activity is incompletely described. Although a number of molecules have been identified that can respond to stimuli, evidence that these molecules can transduce stimulation into useful neural activity is lacking. Here we demonstrate that pickpocket1 (ppk1), a Drosophila homolog of mammalian Degenerin/epithelial sodium channels, encodes an acid-sensing sodium channel that conducts a transient depolarizing current in multidendritic sensory neurons of Drosophila melanogaster. Stimulation of Ppk1 is sufficient to bring these sensory neurons to threshold, eliciting a burst of action potentials. The transient nature of the neural activity produced by Ppk1 activation is the result of Ppk1 channel gating properties. This model is supported by the observation of enhanced bursting activity in neurons expressing a gain of function ppk1 mutant harboring the degenerin mutation. These findings demonstrate that Ppk1 can function as an ionotropic molecular sensory transducer capable of transforming the perception of a stimulus into phasic neuronal activity in sensory neurons.

Tryptophan residues critical to function are frequently located at the lipid-water interface of transmembrane domains. All members of the epithelial Na+ channel (ENaC)/Degenerin (Deg) channel superfamily contain an absolutely conserved Trp at the base of their first transmembrane domain. Here, we test the importance of this conserved Trp to ENaC/Deg function. Targeted substitution of this Trp in mouse ENaC and rat ASIC subunits decrease channel activity. Differential substitution with distinct amino acids in alpha-mENaC shows that it is loss of this critical Trp rather than introduction of residues having novel properties that changes channel activity. Surprisingly, Trp substitution unmasks voltage sensitivity. Mutant ENaC has increased steady-state activity at hyperpolarizing compared with depolarizing potentials associated with transient activation and deactivation times, respectively. The times of activation and deactivation change 1 ms/mV in a linear manner with rising and decreasing slopes, respectively. Increases in macroscopic currents at hyperpolarizing potentials results from a voltage-dependent increase in open probability. Voltage sensitivity is not influenced by divalent cations; however, it is Na+-dependent with a 63-mV decrease in voltage required to reach half-maximal activity per log increase in [Na+]. Mutant channels are particularly sensitive to intracellular [Na+] for removing this sodium abolishes voltage dependence. We conclude that the conserved Trp at the base of TM1 in ENaC/Deg channels protects against voltage by masking an inhibitory allosteric or pore block mechanism, which decreases activity in response to intracellular Na+.

Motor coordination is broadly divided into gross and fine motor control, both of which depend on proprioceptive organs. However, the channels that function specifically in fine motor control are unknown. Here we show that mutations in trpγ disrupt fine motor control while leaving gross motor proficiency intact. The mutants are unable to coordinate precise leg movements during walking, and are ineffective in traversing large gaps due to an inability in making subtle postural adaptations that are requisite for this task. TRPγ is expressed in proprioceptive organs, and is required in both neurons and glia for gap crossing. We expressed TRPγ in vitro, and found that its activity is promoted by membrane stretch. A mutation eliminating the Na(+)/Ca(2+) exchanger suppresses the gap-crossing phenotype of trpγ flies. Our findings indicate that TRPγ contributes to fine motor control through mechanical activation in proprioceptive organs, thereby promoting Ca(2+) influx, which is required for function.

Discretionary control of Na(+) excretion is a key component of the regulation of arterial blood pressure in mammals. Sodium excretion is fine-tuned in the aldosterone-sensitive distal nephron by the activity of the epithelial Na(+) channel (ENaC). Here, ENaC functions as a final effector of the renin-angiotensin-aldosterone system (RAAS) during negative feedback control of blood pressure. Mutations affecting ENaC activity and abnormal regulation of this channel affect blood pressure through pathological changes to Na(+) excretion. Recent evidence demonstrates that powerful signalling pathways function in parallel with the RAAS to modulate ENaC activity and blood pressure. An inclusive paradigm is emerging with respect to regulation of blood pressure where ENaC serves as a critical point of convergence for several important signalling systems that affect renal Na(+) excretion. A robust inhibitory purinergic signalling system intrinsic to the distal nephron dynamically regulates ENaC through paracrine ATP signalling via the metabotropic P2Y2 purinergic receptor to properly match urinary Na(+) excretion to dietary Na(+) intake. This enables blood pressure to be maintained within a normal range despite broad changes in dietary Na(+) consumption. Loss of purinergic inhibition of ENaC increases blood pressure by causing inappropriate Na(+) excretion. In contrast, stimulation of the P2Y2 receptor promotes natriuresis and a decrease in blood pressure. Such observations identify purinergic signalling in the distal nephron as possibly causative, when dysfunctional, for certain forms of elevated blood pressure, and as a possible therapeutic target for the treatment of elevated blood pressure particularly that associated with salt sensitivity.

Adenosine, a purine nucleoside, is present at high concentrations in tumors, where it contributes to the failure of immune cells to eliminate cancer cells. The mechanisms responsible for the immunosuppressive properties of adenosine are not fully understood. We tested the hypothesis that adenosine’s immunosuppressive functions in human T lymphocytes are in part mediated via modulation of ion channels. The activity of T lymphocytes relies on ion channels. KCa3.1 and Kv1.3 channels control cytokine release and, together with TRPM7, regulate T cell motility. Adenosine selectively inhibited KCa3.1, but not Kv1.3 and TRPM7, in activated human T cells. This effect of adenosine was mainly mediated by A2A receptors, as KCa3.1 inhibition was reversed by SCH58261 (selective A2A receptor antagonist), but not by MRS1754 (A2B receptor antagonist), and it was mimicked by the A2A receptor agonist CGS21680. Furthermore, it was mediated by the cAMP/protein kinase A isoform (PKAI) signaling pathway, as adenylyl-cyclase and PKAI inhibition prevented adenosine effect on KCa3.1. The functional implication of the effect of adenosine on KCa3.1 was determined by measuring T cell motility on ICAM-1 surfaces. Adenosine and CGS21680 inhibited T cell migration. Comparable effects were obtained by KCa3.1 blockade with TRAM-34. Furthermore, the effect of adenosine on cell migration was abolished by pre-exposure to TRAM-34. Additionally, adenosine suppresses IL-2 secretion via KCa3.1 inhibition. Our data indicate that adenosine inhibits KCa3.1 in human T cells via A2A receptor and PKAI, thereby resulting in decreased T cell motility and cytokine release. This mechanism is likely to contribute to decreased immune surveillance in solid tumors.

Electrophysiological studies provide essential clues about the regulation and physiological function of ion channel proteins. Probing ion channel activity in vivo, though, often is challenging. This can limit the usefulness of such model organisms as Drosophila for electrophysiological studies. This is unfortunate because these genetically tractable organisms represent powerful research tools that facilitate elaboration of complex questions of physiology. Here, we describe a recently developed method for recording ion channel activity in Drosophila sensory neurons. This approach is based on patch-clamping primary neuron cultures from Drosophila embryos. Such cultures allow the study of ion channels in different genetic backgrounds. In addition to describing how to prepare a primary neuronal cell culture from Drosophila embryos, we discuss, as an example of utility, analysis of Na(+) currents in cultured class IV multidendritic (md) sensory neurons with the patch clamp technique. Excitability of md sensory neurons, manifested as action potential firing, is revealed with whole-cell current-clamping. Voltage-clamping class IV md neurons revealed the activity of the voltage-gated Na(+) channel, paralytic. Moreover, challenging class IV md neurons with acidic pH activates acid-sensing inward Na(+) currents. Genetic manipulation of Drosophila combined with this electrophysiological readout of activity identifies pickpocket1 (Ppk1), a member of the Deg/ENaC channel family, as responsible for conducting an acid-sensing Na(+) current in class IV md sensory neurons.

Electrophysiological studies provide essential clues about the regulation and physiological function of ion channel proteins. Probing ion channel activity in vivo, though, often is challenging. This can limit the usefulness of such model organisms as Drosophila for electrophysiological studies. This is unfortunate because these genetically tractable organisms represent powerful research tools that facilitate elaboration of complex questions of physiology. Here, we describe a recently developed method for recording ion channel activity in Drosophila sensory neurons. This approach is based on patch-clamping primary neuron cultures from Drosophila embryos. Such cultures allow the study of ion channels in different genetic backgrounds. In addition to describing how to prepare a primary neuronal cell culture from Drosophila embryos, we discuss, as an example of utility, analysis of Na(+) currents in cultured class IV multidendritic (md) sensory neurons with the patch clamp technique. Excitability of md sensory neurons, manifested as action potential firing, is revealed with whole-cell current-clamping. Voltage-clamping class IV md neurons revealed the activity of the voltage-gated Na(+) channel, paralytic. Moreover, challenging class IV md neurons with acidic pH activates acid-sensing inward Na(+) currents. Genetic manipulation of Drosophila combined with this electrophysiological readout of activity identifies pickpocket1 (Ppk1), a member of the Deg/ENaC channel family, as responsible for conducting an acid-sensing Na(+) current in class IV md sensory neurons.

Here we explore the mechanism and associated structure-function implications of loss of function for epithelial Na+ channel (ENaC) containing a pseudohypoaldosteronism type 1 (PHA-1)-causing missense point mutation. As expected, human ENaC that contained subunits harboring PHA-1-causing substitutions within an absolutely conserved, cytosolic Gly residue (e.g., βG37S) had significantly less activity. Unexpectedly, though, such substitution also results in voltage sensitivity with greater activity at hyperpolarizing potentials. This is a consequence of voltage-dependent changes in the single-channel open probability and is not species- or subunit-dependent. Voltage sensitivity in PHA-1 mutants stems from the disruption of critical structure, rather than the development of new properties resulting from the introduction of novel side chains. Residues near the conserved His-Gly sequence of G95 in α-mENaC are particularly important for voltage sensing. Although substitution of I93 in α-mENaC results in voltage sensing, it also slows the activation and deactivation kinetics enough to enable capture of the dynamic changes in single-channel open probability that account for changes in macroscopic activity. This provides definitive proof of the mechanism that underlies loss of function. In addition, the voltage dependence of ENaC with PHA-1 substitutions is akin to that which results from substitution of a critical, interfacial Trp residue conserved at the intracellular base of TM1 (e.g., W112 in α-mENaC). Dynamic interactions between similarly positioned His and Trp residues are essential for gating and the girdle-like structure that lines the intracellular mouth of the M2 proton channel. The similar residues in ENaC may serve a shared function, suggesting the possibility of an intracellular girdle just below the mouth of the ENaC pore.

These studies test whether three disease‐causing mutations in genes ( SCNN1A and SCNN1G ) encoding subunits of the epithelial Na + channel, ENaC, affect the biophysical and gating properties of this important renal ion channel. The S562P missense mutation in α ENaC and the K106_S108delinsN mutation in γ ENaC are associated with pseudohypoaldosteronism type 1 (PHA1). The N530S missense mutation in γ ENaC causes Liddle9s syndrome. Incorporation of S562P into α ENaC and K106_S108N into γ ENaC resulted in significant decreases in macroscopic ENaC currents. Conversely, incorporation of N530S into γ ENaC increased macroscopic ENaC current. The S562P substitution resulted in a nonfunctional channel. The K106_S108N mutation produced a functional channel having a normal macroscopic current–voltage relation, there was a slight but significant decrease in unitary conductance and a marked decrease in single‐channel open probability. The N530S substitution increased single‐channel open probability having no effect on the macroscopic current–voltage relation or unitary conductance of the channel. These findings are consistent with mutation of residues at 562 in α ENaC and 530 in γ ENaC, and a 3′ splice site in SCNN1G (318‐1 G→A; K106_108SdelinsN) resulting in aberrant ENaC activity due to changes in the biophysical and gating properties of the channel. Such changes likely contribute to the cellular mechanism underpinning the PHA1 and Liddle9s syndrome caused by these mutations in ENaC subunits.