Native Channels Research Articles

Phospholipase D2 Specifically Regulates TREK Channels via Direct Interaction and Local Production of Phosphatidic Acid

Membrane lipids serve as second messengers and docking sites for proteins and play central roles in cell signalling. A major question about lipid signaling is whether diffusible lipids can selectively target specific proteins. One family of lipid-regulated membrane proteins is the TREK subfamily of K2P channels: TREK1, TREK2, and TRAAK. We investigated the regulation of TREK channels by phosphatidic acid (PA), which is generated by Phospholipase D (PLD) via hydrolysis of phosphatidylcholine. We found that, even though all three of the channels are sensitive to PA, only TREK1 and TREK2 are potentiated by PLD2 and that none of these channels is modulated by PLD1, indicating surprising selectivity. We find that PLD2, but not PLD1, directly binds to the C-terminus of TREK1 and TREK2, but not to TRAAK. The results lead to a model for selective lipid regulation by localization of phospholipid enzymes to specific effector proteins. Finally, by using the photoswitchable conditionnal subunit method to endow light sensitivity to the native TREK1 channels, we show that regulation of TREK channels by PLD2 occurs natively in hippocampal neurons.

Inhibition of T-type Ca2+ Channels by Hydrogen Sulfide.

T-type Ca(2+) channels are a distinct family of low voltage-activated Ca(2+) channels which serve many roles in different tissues. Several studies have implicated them, for example, in the adaptive responses to chronic hypoxia in the cardiovascular and endocrine systems. Hydrogen sulfide (H(2)S) was more recently discovered as an important signalling molecule involved in many functions, including O(2) sensing. Since ion channels are emerging as an important family of target proteins for modulation by H(2)S, and both T-type Ca(2+) channels and H(2)S are involved in cellular responses to hypoxia, we have investigated whether recombinant and native T-type Ca(2+) channels are a target for modulation by H(2)S. Using patch-clamp electrophysiology, we demonstrate that the H(2)S donor, NaHS, selectively inhibits Cav3.2 T-type Ca(2+) channels heterologously expressed in HEK293 cells, whilst Cav3.1 and Cav3.3 channels were unaffected. Sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn(2+) to this channel. Chelation of Zn(2+) using TPEN prevented channel inhibition by H(2)S. H2S also selectively inhibited native T-type channels (primarily Cav3.2) in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H(2)S regulation, the T-type Ca(2+) channel Cav3.2. Results have important implications for the proposed pro-nociceptive effects of this gasotransmitter. Implications for the control of cellular responses to hypoxia await further study.

Effect of Ligand Binding to the B1B Subunit of Olfactory CNG Channels

Cyclic nucleotide-gated (CNG) channels play an essential role in the sensory transduction of the olfactory and visual system. Native olfactory CNG channels are heterotetramers composed of three different subunits: two CNGA2, one CNGA4 and one CNGB1b. It is well established that the presence of the CNGA4 and CNGB1b subunit in heterotetrameric channels leads to an increased apparent affinity for cyclic nucleotides. We have recently shown that the CNGA4 subunit binds cGMP and actively participates to channel activation. Nevertheless, the functional contribution of the CNGB1b subunit is still unclear. By combining mutagenesis and confocal patch-clamp fluorometry we characterized the role of the CNGB1b subunit during channel activation. We show that in contrast to the CNGA4 subunit, that needs only neighbouring CNGA2 subunits for ligand binding, the CNGB1b needs both, CNGA2 and CNGA4. When coexpressing CNGB1b with disabled CNGA2 and CNGA4, channel activation induced by the CNGB1b subunit alone was only weak compared to CNGA4 coexpressed with disabled CNGA2. The apparent affinity (EC50) of the CNGB1b subunit under the same conditions is 3.2 µM. This is twofold higher than that determined for CNGA4 and 15 fold higher than that of one CNGA2. The binding of cAMP and cGMP to the CNGB1b subunit turned out to be non-selective. Moreover, the kinetics of ligand binding and unbinding as well as channel activation and deactivation for each of the three different subunits in the respective environment of disabled subunits, substantiate the role of the CNGB1b and CNGA4 in speeding up channel closure. In conclusion, we show that olfactory CNG channels are activated by ligand binding to all four subunits, but the contribution of the CNGB1b to channel activation is only modest in comparison with that of the other subunits.

Studying the Influence of the Subunit Arrangement on the Function of Heterotetrameric Olfactory CNG Channels

Cyclic nucleotide-gated (CNG) ion channels, key components in the sensory transduction of vision and odour, are non-selective cation channels, activated by the binding of cAMP or cGMP. Native CNG channels of olfactory sensory neurons are heterotetramers formed by the homologous CNGA2, CNGA4 and CNGB1b subunits in a 2:1:1 stoichiometry. It is still an unsolved question how the individual subunits are arranged with respect to each other.Addressing this, we used a concatenation approach to force the heterotetrameric channels to a defined order of the subunits. We designed all twelve possible concatamers representing three possibilities of arrangement for the monomers around a central pore. Ligand-dependent activation induced by the binding of either cAMP or cGMP was characterized electrophysiologically for all concatamers in macropatches of Xenopus laevis oocytes as well as on the single-channel level. All twelve concatamers formed functional channels with robust expression levels comparable to monomeric channels. As for channels built of monomers, Hill coefficients for cGMP exceeded those for cAMP (1.8 versus 1.5), but were equal for the same ligand with all concatamers. The EC50 values were moderately different among the concatamers without revealing obvious systematics. B1bA2A4A2 proved to be the most and A2A2B1bA4 the least ligand-sensitive channel. Like for channels built of monomers, all twelve concatamers exhibited a typical and complex single-channel behavior with a main single-channel conductance of about 12 pS and 15 pS at positive and negative voltages, respectively.We conclude that all possible arrangements of the three types of subunits lead to functional heterotetrameric channels with similar properties, suggesting that essential functional specificities inferred by the individual subunits do not critically depend on the nature of their specific neighbor subunits.

β-2 and γ-1 Auxiliary Subunits Coassemble in the Same BK Channel and Independently Contribute to Regulation of Channel Gating

Large conductance, Ca2+-and-voltage activated K+ (BK) channels are oligomeric proteins whose minimal functional unit is a homotetramer of the pore-forming α subunit. The α subunit is almost ubiquitously expressed in mammalian tissues, but native BK channel complexes can also contain associated auxiliary subunits which are tissue-specifically expressed. Members of two structurally distinct families of auxiliary subunits, β1- β4 and γ1-γ3, have each been shown to separately assemble with BK-α subunit. Each auxiliary subunit defines important gating properties of the BK channel complex as well as its pharmacology. However, the possibility of coassembly of the two types of auxiliary subunits in the same BK channel has not been closely examined. The only antecedent reports that overexpression of β1 subunit precludes the ability of γ1 to affect BK gating. Here, using single channel and macroscopic patch-clamp recordings we show that β2 and γ1 can coassemble in the same channel. When α, β2 and γ1 subunits are coexpressed at typical RNA ratios, the resulting BK-current shows a complete inactivation, indicating the presence of β2 in all channels, but also exhibits a gating shift consistent with the presence of γ1 in all channels. The effects produced by β2 and γ1 subunits on BK channel gating are approximately additive, consistent with independent mechanistic effects. Furthermore, coassembly of both subunits in the same channel produces a constitutive inactivation of BK current at physiological conditions even at 0 [Ca2+]. This possibility that β and γ subunits may also coassemble in the same BK channels in native tissues adds significantly to the potential functional diversity of BK channels.This work was supported by National Institutes of Health Grant GM-081748 (to C.J.L.) and National Research Service Award GM103138 (to V.G.-P.).

Studying the Gating Mechanism of Mammalian Kir Channels using in Silico Mutations

Inward rectifier potassium (Kir) channels are essential membrane proteins, controlling the permeation of K+ ions across membranes. They are found in eukaryotes as well as bacteria. These channels are expressed in multiple tissues and responsible for a wide range of physiological processes i.e. shaping the action potential, insulin decrease and neurotransmitter response. Crystal structures have provided major insights into the architecture of these channels. So far, all mammalian structures have been captured in a closed state. Recently, the first putative open structure of a bacterial Kir channel was obtained using a gating mutant (S129R) close to the helix bundle crossing region (HBC) (Bavro et al., 2012). Thus, we reasoned that introducing such a mutant in a mammalian Kir channel might enable investigating the open state.Therefore, we introduced in silico mutations in all for subunits at gating sensitive regions and conducted up to 1μs molecular dynamics simulations.Intriguingly, during the mutant simulations major conformational changes were observed. In all simulations, widening at the HBC gate occurred. These changes enabled continuous water flux through this gate and access for hydrated potassium ions to the cavity. A major difference to native mammalian Kir channels is the PIP2 dependence. In contrast to the wild type channels, our charged mutant channels do not need PIP2 to induce opening. This is likely due to the strong repulsive effects of the negative charges. Importantly, changes in the lipid interactions of the slide helix reveal a movement of this helix towards the lipid head groups during opening, in agreement with experimentally observed lipid interaction changes (Lee et al., 2013).It cannot be excluded that mutant channels stabilize rarely populated open states, as previously reported for selected bacterial Kir channel mutants (Zubcevic et al. 2014).

Continent catheterizable conduit for urinary diversion in children: Applicability and acceptability.

Background:Continent catheterizable conduit (CCC) has made clean intermittent catheterization (CIC) painless and easy. It is applicable in diverse clinical conditions. Nonetheless, convincing the parents for the need of conduit procedure is still difficult.Materials and Methods:A prospective study, included children who underwent CCC procedure from March 2008 to February 2013. The data were assessed for; diagnosis, type of conduit, number of preoperative counselling sessions before acceptance, role of “self-help group” in decision making, parental concern and satisfaction for the procedure.Results:Twenty-nine patients (males; 24, females; 5) underwent CCC procedure for various clinical conditions. The multiple preoperative counselling sessions and creation of “self-help groups” were helped them for decision making. The main concerns among parents were: (1) Impact of procedure on future fertility and sexual life. (2) Patency of native urethral channel. (3) Permanent urinary stoma over the abdomen.Conclusion:CCC procedures are applicable to a wide array of clinical situations with a good outcome. The acceptability of the CCC procedure improves with preoperative counselling of parent/child, initiation of preoperative per urethral CIC and creation of self-help groups.

STIM1 and STIM2 Proteins Regulation of Endogenous Store-Operated Calcium Channels in HEK293 Cells

The endoplasmic reticulum calcium sensors STIM1 and STIM2 are key modulators of store-operated calcium entry. They play a major role in physiological functions in the norm and pathology, however, available data on native STIM2-regulated plasma membrane channels are scarce: it is in only a few studies that STIM2-induced CRAC currents have been recorded. The STIM1 protein has been shown to regulate not only CRAC but also TRPC channels, but it has remained unclear whether STIM2 is capable of regulating store-operated non-CRAC channels. Here we present experimental evidence for the existence of endogenous non-CRAC STIM2-regulated channels. In single-channel patch-clamp experiments on HEK293 cells, store-operated Imin channels were induced by selective activation of native STIM2 proteins or STIM2 overexpression. It was found that STIM1 activation blocked store-operated mode of Imin activation. Changes in the ratio between active STIM2 and STIM1 proteins could switch Imin channels regulation between store-operated and store-independent modes.We have previously characterized electrophysiological properties of different Ca2+ influx channels coexisting in HEK293 cells. The results of this study show that STIM1 and STIM2 differ in the ability to activate these store-operated channels: Imin channels are regulated by STIM2, TRPC3-containing INS channels are induced by STIM1, and TRPC1-composed Imax channels are activated by both STIM1 and STIM2. These new data about cross-talk between STIM1 and STIM2 and their different roles in store-operated channel activation are indicative of an additional level in the regulation of store-operated calcium entry pathways.This study was supported by the Russian Scientific Foundation 14-14-00720 (to E.K. and A.S.); the program “Molecular and Cellular Biology” of the RAS (to G.N.M and L.G.); the Scientific School Support Program SS-1721.2014.4 (to G.N.M.); the Dynasty Foundation; and the Russian Foundation for Basic Research.

SK Channel Distribution on Neuronal Axons Revealed by Single Molecule Atomic Force Microscopy

Introduction: The axon initial segment (AIS) is a specialized cellular compartment where action potentials (APs) initiate. Calcium-activated small conductance potassium (SK) channels reduce excitability by mediating the afterhyperpolarizing (AHP) current and thus modulate the intervals between spikes during a burst of action potentials. SK channels have a polarized distribution in neurons with high density in dendrites and lower density in the soma. However, it is not clear if and which SK channels are expressed on the axon and their spatial distribution. Here, we employ single molecule atomic force microscopy (AFM) combined with natural toxins to determine the localization of SK channels on neuronal axons.Methods and Results: We have previously demonstrated that integration of single molecule AFM and toxin pharmacology allows for high resolution mapping of native SK2 channels on soma and dendrites of living neurons. AFM tip functionalized with SK channel blocker-apamin, was used to detect the distribution of SK channels by measuring the unbinding forces between apamin and SK channels. The axon was identified by transfecting rat pyramidal neurons with tau-gfp which localizes at the axon. Apamin-functionalized AFM tips were used to probe 1µm∧2 scan area on the axon of transfected pyramidal neurons. We detected unbinding forces with a mean of µ=20± 9pN and a surface density of 5.9% (n=8). To determine the specificity of the detected unbinding forces, the experiments were repeated with neurons pretreated with apamin. Along with a lower frequency of unbinding events, we found that in neurons pretreated with apamin, there was a decrease in the unbinding forces. The results above indicate that apamin sensitive SK channels reside on the axon of pyramidal neurons.Conclusion: Employing single molecule AFM, we have revealed that SK channels reside on axonal surfaces of neurons.

Molecular Dynamics of Selective Sugar Permeation through the Connexin26 Channel

A signal property of connexin channels is the ability to mediate selective diffusive movement of molecules through plasma membrane, yet the movement of biological molecules through these channels has yet to be well-characterized in mechanistic or energetic terms. Different connexin channels have distinct molecular selectivities that cannot be explained on the basis of size or charge of the permeants; the forces that molecules experience within the pore determines which molecules are permeable and to what degree. The energetics of the movement of two derivatized sugars, one permeable and one impermeable, through an experimentally validated connexin26 (Cx26) structural model were explored using Hamiltonian Replica Exchange MD Umbrella Sampling (US/H-REMD) and Steered Molecular Dynamics (SMD), and associated analytic tools. Crucially, the Cx26 channel model, in explicit membrane/solvent, incorporates key post-translational charge changes shown by Brownian Dynamics to be required to reproduce the electrical conductance characteristics of the native channel [Kwon J.Gen.Physiol. 138:4751]. The results show energy profiles consistent with experimental results. The energetic barriers extend through most of the pore length, rather than being highly localized, as in ion-specific channels. There is little evidence for binding within the pore. Force decomposition reveals how, for each test molecule, interactions with water and with the Cx26 protein vary over the length of the pore, and reveal a significant contribution of interaction with K+ ions. The flexibility of pore width varies along its length, and the test molecules tend to widen the pore as they pass through. This work highlights factors involved in selective molecular permeation that may not be significant for atomic ions. The results suggest that this system can be used to explore the molecular basis by which connexin channels select among (potential) permeating molecules, and how mutations alter the permeation process.

A sustained increase in the intracellular Ca2+ concentration induces proteolytic cleavage of EAG2 channel

Voltage-gated EAG2 channel is abundant in the brain and enhances cancer cell growth by controlling cell volume. The channel contains a cyclic nucleotide-binding homology (CNBH) domain and multiple calmodulin-binding motifs. Here we show that a raised intracellular Ca2+ concentration causes proteolytic digestion of heterologously expressed and native EAG2 channels. A treatment of EAG2-expressing cells with the Ca2+ ionophore A23187 for 1h reduces the full-length protein by ∼80% with a concomitant appearance of 30–35-kDa peptides. Similarly, a treatment with the Ca2+-ATPase inhibitor thapsigargin for 3h removes 30–35-kDa peptides from ∼1/3 of the channel protein. Moreover, an incubation of the isolated rat brain membrane with CaCl2 leads to the generation of fragments with similar sizes. This Ca2+-induced digestion is not seen with EAG1. Mutations in a C-terminal calmodulin-binding motif alter the degrees and positions of the cleavage. Truncated channels that mimic the digested proteins exhibit a reduced current density and altered channel gating. In particular, these shorter channels lack a rapid activation typical in EAG channels with more than 20-mV positive shifts in voltage dependence of activation. The truncation also eliminates the ability of EAG2 channel to reduce cell volume. These results suggest that a sustained increase in the intracellular Ca2+ concentration leads to proteolytic cleavage at the C-terminal cytosolic region following the CNBH domain by altering its interaction with calmodulin. The observed Ca2+-induced proteolytic cleavage of EAG2 channel may act as an adaptive response under physiological and/or pathological conditions.

Actions of the pyrethroid insecticide bifenthrin on sodium channels expressed in rat cerebral cortical neurons

Voltage-gated sodium channels are important sites for the neurotoxic actions of pyrethroid insecticides in mammals. Here, we studied the mode of action of bifenthrin on the native sodium channels in cerebral cortical neurons prepared from newborn rat brain, where the toxic effects are largely generated. Bifenthrin caused a pronounced late current that persisted at the end of a depolarizing pulse, a slowly-decaying tail current following repolarization and significant resting modification (25.3% modification at 10 μM). No significant bifenthrin-induced effect was observed at the peak current. Bifenthrin also caused a concentration-dependent hyperpolarizing shift in steady-state activation and inactivation as well as slowed recovery from channel inactivation. Repetitive depolarization increased the potency of bifenthrin with high frequency. There was approximately 64% inhibition of modification upon repetitive activation by 10-Hz trains of depolarizing pulses. These results suggest that bifenthrin binds to and modifies sodium channels in both the closed and open states and exhibits the behavior between type I and type II.

Preferential assembly of heteromeric small conductance calcium-activated potassium channels.

The activation of small conductance calcium-dependent (SK) channels regulates membrane excitability by causing membrane hyperpolarization. Three subtypes (SK1-3) have been cloned, with each subtype expressed within the nervous system. The locations of channel subunits overlap, with SK1 and SK2 subunits often expressed in the same brain region. We showed that expressed homomeric rat SK1 subunits did not form functional channels, because subunits accumulated in the Golgi. This raised the question of whether heteromeric channels could form with SK1 subunits. The co-expression of SK1 and SK2 subunits in HEK293 cells preferentially co-assembled to produce heteromeric channels with a fixed stoichiometry of alternating subunits. The expression in hippocampal CA1 neurons of mutant rat SK1 subunits [rat SK1(LV213/4YA)] that produced an apamin-sensitive current changed the amplitude and pharmacology of the medium afterhyperpolarization. The overexpression of rat SK1(LV213/4YA) subunits reduced the sensitivity of the medium afterhyperpolarization to apamin, substantiating the preferential co-assembly of SK1 and SK2 subunits to form heteromeric channels. Species-specific channel assembly occurred as the co-expression of human SK1 with rat SK2 did not form functional heteromeric channels. The replacement of two amino acids within the C-terminus of rat SK2 with those from human SK2 permitted the assembly of heteromeric channels when co-expressed with human SK1. These data showed that species-specific co-assembly was mediated by interaction between the C-termini of SK channel subunits. The finding that SK channels preferentially co-assembled to form heteromeric channels suggested that native heteromeric channels will predominate in cells expressing multiple SK channel subunits.

Inhibition of acid-sensing ion channels by levo-tetrahydropalmatine in rat dorsal root ganglion neurons.

Levo-tetrahydropalmatine (l-THP), a main bioactive Chinese herbal constituent from the genera Stephania and Corydalis, has been in use in clinical practice for years in China as a traditional analgesic agent. However, the mechanism underlying the analgesic action of l-THP is poorly understood. This study shows that l-THP can exert an inhibitory effect on the functional activity of native acid-sensing ion channels (ASICs), which are believed to mediate pain caused by extracellular acidification. l-THP dose dependently decreased the amplitude of proton-gated currents mediated by ASICs in rat dorsal root ganglion (DRG) neurons. l-THP shifted the proton concentration-response curve downward, with a decrease of 40.93% ± 8.45% in the maximum current response to protons, with no significant change in the pH0.5 value. Moreover, l-THP can alter the membrane excitability of rat DRG neurons to acid stimuli. It significantly decreased the number of action potentials and the amplitude of the depolarization induced by an extracellular pH drop. Finally, peripherally administered l-THP inhibited the nociceptive response to intraplantar injection of acetic acid in rats. These results indicate that l-THP can inhibit the functional activity of ASICs in dissociated primary sensory neurons and relieve acidosis-evoked pain in vivo, which for the first time provides a novel peripheral mechanism underlying the analgesic action of l-THP.

21.: Carbamazepine inhibition of native sodium currents in murine osteoblasts

An increase in fracture risk is well established in patients with epilepsy. However, the mechanism for this association is less well defined. Our aim was to isolate sodium channel currents in mouse primary calvarial osteoblasts using whole-cell patch-clamping and also to examine whether the anti-epileptic medication carbamazepine (CBZ) blocks these currents. Primary osteoblasts isolated from the calvariae of C57BL6 mice at postnatal day 2 (P2) were used to examine the impact of CBZ on sodium channel currents using automated whole-cell planar electrode voltage clamp recordings (Patchliner, Nanion, Germany). Currents were elicited from a holding potential of −80mV using a voltage protocol stepping from −100mV to +60mV in 10mV increments, for 20ms. CBZ (50μM) was applied to the cells in the continued presence of external tetraethylammonium (10mM) and internal Cs⁺. Following washout of CBZ, 10μM tetrodotoxin (TTX), a known voltage-gated sodium channel blocker, was applied. TTX sensitive (mean block of 89.96±2.14%; n=6; <i>p</i>< 0.0001) voltage-activated inward sodium currents were readily observed in P2 osteoblasts and bath application of CBZ (50μM) significantly inhibited these currents (mean block of 31.6±5.9%; n=7; <i>p</i><0.001). Mouse osteoblasts express native voltage-activated sodium channels, which are sensitive to CBZ and TTX. This novel demonstration of an inhibitory effect of CBZ on primary osteoblast sodium currents utilising Patchliner paves the way for semi-automated screening of anti-epileptic drugs, enabling further study to determine whether the effects on ion channel activity observed here translate to effects on signal transduction in osteoblasts, inhibition of osteoblast activity and clinically-relevant changes in bone quality.

Double Channel Pylorus as a Complication of Peptic Ulcer Disease

Introduction: Double channel pylorus is a rare condition consisting of a gastro duodenal fistula extending from the gastric antrum to the duodenal bulb through an accessory canal. The double pylorus is a destructive but relatively benign complication of peptic ulcer disease. It could be congenital or acquired. Case Report: A 73-year-old female with history of cirrhosis secondary to non-alcoholic steatohepatitis with splenomegaly, thrombocytopenia, and peptic ulcer disease was admitted to the hospital for progressive shortness of breath and noted to have iron deficiency anemia. The patient underwent screening upper endoscopy as a part of screening for esophageal varices. She was noted to have some antral thickening extending to pylorus. Biopsies showed that the thickening was only focal fibrosis and reactive glandular atypia, but no tumor. Six months later, the patient was readmitted with worsening shortness of breath, and was found to have anemia and positive occult blood. Colonoscopy was done as a part of work-up, which was completely normal. Later, the patient underwent esophagogastroduodenoscopy (EGD). On EGD, the changes were consistent with mild gastritis and a duplicate opening lying adjacent to pyloric channel. The scope was passed through both the openings. First was the native pyloric channel. The duplicated pyloric channel seemed to open into the second part of the duodenum, and there was a large, clean bases ulcer, but no fistulas. There was shelve of normal tissue diving the 2 openings. Upper GI series was also done to confirm the diagnosis. Discussion: The prevalence of double pylorus varies from 0.06 to 0.4%; however, its real incidence remains unknown. It occurs more often in men (2:1), as well as with other peptic diseases. Malignant tumors resulting in formation of double pylorus is extremely rare. Clinical symptoms are similar to those of peptic ulcer disease. They are identified on upper endoscopy and can also be seen on barium meal study. Convergence of double pyloric canals resulting into 1 large gastrointestinal passage can predispose to bile reflux. Other complications, like hemorrhage or intractable pain, may require surgical intervention. Double channel pylorus is a rare but important complication of peptic ulcer disease, and should be identified early to prevent complications.

Classical Transient Receptor Potential 1 (TRPC1): Channel or Channel Regulator?

In contrast to other Classical Transient Receptor Potential TRPC channels the function of TRPC1 as an ion channel is a matter of debate, because it is often difficult to obtain substantial functional signals over background in response to over-expression of TRPC1 alone. Along these lines, heterologously expressed TRPC1 is poorly translocated to the plasma membrane as a homotetramer and may not function on its own physiologically, but may rather be an important linker and regulator protein in heteromeric TRPC channel tetramers. However, due to the lack of specific TRPC1 antibodies able to detect native TRPC1 channels in primary cells, identification of functional TRPC1 containing heteromeric TRPC channel complexes in the plasma membrane is still challenging. Moreover, an extended TRPC1 cDNA, which was recently discovered, may seriously question results obtained in heterologous expression systems transfected with shortened cDNA versions. Therefore, this review will focus on the current status of research on TRPC1 function obtained in primary cells and a TRPC1-deficient mouse model.

Phenylarsine oxide as a redox modulator of transient receptor potential vanilloid type 1 channel function.

Transient receptor potential vanilloid type 1 (TRPV1) channels are capable of detecting and integrating noxious stimuli and play an important role in nociceptor activation and sensitization. It has been demonstrated that oxidizing agents are capable of positively modulating (sensitizing) the TRPV1 channel. The present study investigates the ability of the thiol-oxidizing agent phenylarsine oxide (PAO) to modulate TRPV1 currents under voltage-clamp conditions. We assessed the ability of PAO to modulate both proton- and capsaicin-activated currents mediated by recombinant human TRPV1 channels as well as native rat and human TRPV1 channels in dorsal root ganglion (DRG) neurons. Experiments with other oxidizing and reducing agents having various membrane-permeating properties supported the intracellular oxidizing mechanism of PAO modulation. The PAO modulation of proton-activated currents was consistent across the cell types studied, with an increase in current across the proton concentrations studied. PAO modulation of the capsaicin-activated current in hTRPV1/Chinese hamster ovary cells consisted of potentiation of the current elicited with low capsaicin concentrations and inhibition of the current at higher concentrations. This same effect was seen with these recombinant cells in calcium imaging experiments and with native TRPV1 channels in rat DRG neurons. Contrary to this, currents in human DRG neurons were potentiated at all capsaicin concentrations tested after PAO treatment. These results could indicate important differences in the reduction-oxidation modulation of human TRPV1 channels in a native cellular environment.

Monoamine NMDA receptor channel blockers inhibit and potentiate native and recombinant proton-gated ion channels

Acid-sensing ion channels (ASICs) are widely distributed in the peripheral and central nervous system. Although they are involved in many physiological functions, the actual processes that activate ASICs remain unclear. This is particularly true for brain ASICs, which produce only a transient response to a fast drop in pH and cannot mediate sustained current. Therefore, the search for ASIC inhibitors and, especially, potentiators/activators is important. We report that NMDA receptor channel blockers with a comparatively simple structure (9-aminoacridine, memantine, IEM-2117 and IEM-1921) potentiate and/or inhibit ASICs in submillimolar concentrations. The experiments were performed using the patch clamp technique on native ASICs from rat hippocampal interneurons and recombinant ASICs of different subunit compositions expressed in CHO cells. Native ASICs were potentiated by IEM-1921 and IEM-2117, and inhibited by memantine and 9-aminoacridine. Homomeric ASIC1a were inhibited by memantine, IEM-2117 and 9-aminoacridine while IEM-1921 was ineffective. In contrast, homomeric ASIC2a were potentiated by IEM-2117, memantine and IEM-1921, whereas 9-aminoacridine was inactive. The compounds caused a complex effect on ASIC3. 9-aminoacridine and IEM-1921 potentiated the steady-state response of ASIC3 and inhibited the peak component. IEM-2117 not only potentiated ASIC3-mediated currents caused by acidification but also evoked steady-state currents at neutral pH. Our results demonstrate that, depending on the subunit composition, ASICs can be activated or inhibited by simple compounds that possess only amino group and aromatic/hydrophobic moieties. This opens up the possibility to search for new ASIC modulators among a number of endogenous ligands.

Hydrogen sulfide inhibits Cav3.2 T-type Ca2+ channels.

The importance of H2S as a physiological signaling molecule continues to develop, and ion channels are emerging as a major family of target proteins through which H2S exerts many actions. The purpose of the present study was to investigate its effects on T-type Ca(2+) channels. Using patch-clamp electrophysiology, we demonstrate that the H2S donor, NaHS (10 μM-1 mM) selectively inhibits Cav3.2 T-type channels heterologously expressed in HEK293 cells, whereas Cav3.1 and Cav3.3 channels were unaffected. The sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn(2+) to this channel. Chelation of Zn(2+) with N,N,N',N'-tetra-2-picolylethylenediamine prevented channel inhibition by H2S and also reversed H2S inhibition when applied after H2S exposure, suggesting that H2S may act via increasing the affinity of the channel for extracellular Zn(2+) binding. Inhibition of native T-type channels in 3 cell lines correlated with expression of Cav3.2 and not Cav3.1 channels. Notably, H2S also inhibited native T-type (primarily Cav3.2) channels in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T-type Ca(2+) channel Cav3.2, and suggest that such modulation cannot account for the pronociceptive effects of this gasotransmitter.