Increase In Open Probability Research Articles

Potentiation of BKCa channels by cystic fibrosis transmembrane conductance regulator correctors VX-445 and VX-121.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The large-conductance calcium-activated potassium channel (BKCa, KCa1.1) is also critical for maintaining lung airway surface liquid (ASL) volume. Here, we show that the class 2 (C2) CFTR corrector VX-445 (elexacaftor) induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121, a corrector under clinical evaluation. Whole-cell patch-clamp recordings verified that CFTR correctors potentiated BKCa activity from both primary HBEs and HEK cells stably expressing the α subunit (HEK-BK cells). Furthermore, excised patch-clamp recordings from HEK-BK cells verified direct action on the channel and demonstrated a significant increase in open probability. In mouse mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced firing frequency in primary rat hippocampal and cortical neurons. We raise the possibilities that C2 CFTR correctors gain additional clinical benefit by activation of BKCa in the lung yet may lead to adverse events through BKCa activation elsewhere.

Impact of Mammalian Two-Pore Channel Inhibitors on Long-Distance Electrical Signals in the Characean Macroalga Nitellopsis obtusa and the Early Terrestrial Liverwort Marchantia polymorpha.

Inhibitors of human two-pore channels (TPC1 and TPC2), i.e., verapamil, tetrandrine, and NED-19, are promising medicines used in treatment of serious diseases. In the present study, the impact of these substances on action potentials (APs) and vacuolar channel activity was examined in the aquatic characean algae Nitellopsis obtusa and in the terrestrial liverwort Marchantia polymorpha. In both plant species, verapamil (20–300 µM) caused reduction of AP amplitudes, indicating impaired Ca2+ transport. In N. obtusa, it depolarized the AP excitation threshold and resting potential and prolonged AP duration. In isolated vacuoles of M. polymorpha, verapamil caused a reduction of the open probability of slow vacuolar SV/TPC channels but had almost no effect on K+ channels in the tonoplast of N. obtusa. In both species, tetrandrine (20–100 µM) evoked a pleiotropic effect: reduction of resting potential and AP amplitudes and prolongation of AP repolarization phases, especially in M. polymorpha, but it did not alter vacuolar SV/TPC activity. NED-19 (75 µM) caused both specific and unspecific effects on N. obtusa APs. In M. polymorpha, NED-19 increased the duration of repolarization. However, no inhibition of SV/TPC channels was observed in Marchantia vacuoles, but an increase in open probability and channel flickering. The results indicate an effect on Ca2+ -permeable channels governing plant excitation.

Melatonin activates BKCa channels in cerebral artery myocytes via both direct and MT receptor/PKC-mediated pathway

The pineal hormone melatonin is a neuroendocrine hormone with high membrane permeability that is involved in regulation of circadian rhythm of several biological functions. Large-conductance Ca2+-activated K+ (BKCa) channels are abundantly expressed in vascular smooth muscle cells and play an important role in vascular tone regulation. We investigated the mechanisms through which myocyte BKCa channels mediate effects of melatonin on cerebral arteries (CAs). Arterial contractility measurements showed that melatonin alone did not change vascular tone in CAs; however, it induced concentration-dependent vasodilation of phenylephrine-induced contraction in CAs. In the presence of the potent endothelial oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester, melatonin-elicited relaxation was significantly inhibited by iberiotoxin (BKCa channel blocker). Melatonin significantly increased BKCa currents but not voltage-gated K+ (KV) currents in whole-cell recordings. Melatonin decreased the amplitude of Ca2+ sparks and spontaneous transient outward currents (STOCs), however, a significant increase in open probability of BKCa channels was observed in both inside-out and cell-attached patch-clamp recordings. This melatonin-induced enhancement of BKCa channel activity was significantly suppressed by luzindole (melatonin MT1/MT2 receptor inhibitor), U73122 (phospholipase C (PLC) inhibitor), and Ro31–8220 (protein kinase C (PKC) inhibitor). Melatonin had no significant effects on sarcoplasmic reticulum release of Ca2+. These findings indicate that melatonin-induced vasorelaxation of CAs is partially attributable to direct (passing through the cell membrane) and indirect (via melatonin MT1/MT2 receptors-PLC-PKC pathway) activation of BKCa channels on CA myocytes.

The syndromic deafness mutation G12R impairs fast and slow gating in Cx26 hemichannels.

Mutations in connexin 26 (Cx26) hemichannels can lead to syndromic deafness that affects the cochlea and skin. These mutations lead to gain-of-function hemichannel phenotypes by unknown molecular mechanisms. In this study, we investigate the biophysical properties of the syndromic mutant Cx26G12R (G12R). Unlike wild-type Cx26, G12R macroscopic hemichannel currents do not saturate upon depolarization, and deactivation is faster during hyperpolarization, suggesting that these channels have impaired fast and slow gating. Single G12R hemichannels show a large increase in open probability, and transitions to the subconductance state are rare and short-lived, demonstrating an inoperative fast gating mechanism. Molecular dynamics simulations indicate that G12R causes a displacement of the N terminus toward the cytoplasm, favoring an interaction between R12 in the N terminus and R99 in the intracellular loop. Disruption of this interaction recovers the fast and slow voltage-dependent gating mechanisms. These results suggest that the mechanisms of fast and slow gating in connexin hemichannels are coupled and provide a molecular mechanism for the gain-of-function phenotype displayed by the syndromic G12R mutation.

WS06.2 R117H-CFTR has a defect in channel gating activity that can be potentiated by ivacaftor

Background The R117H mutation is associated with residual chloride transport and a delayed onset of CF symptoms. Although generally called a conductance mutation, R117H-CFTR exhibits defective channel gating. This study aimed to more fully explore the mechanistic defect of R117H-CFTR and to evaluate the effect of ivacaftor, a CFTR potentiator, on R117H-CFTR. Methods and Results In single-channel, patch-clamp studies, the channel open probability (gating activity) of R117H-CFTR was 22% of normal, whereas the channel conductance was 88% of normal. The channel gating activity of R117H-CFTR was characterized by frequent but brief openings of the channel compared with normal CFTR. The brief openings are consistent with a low channel open probability due in part to destabilization of the channel open state by R117H. The addition of ivacaftor increased the channel open probability of R117H-CFTR from 22% to 42% of normal. The increase in open probability was due to an increase in the frequency of channel opening and a decreased closed duration. Channel open duration and conductance were not increased. In Ussing chamber studies using R117H-5T/F508del-human bronchial epithelial cells from two donor bronchi, chloride transport was 23% of normal, which is consistent with residual chloride transport. Ivacaftor treatment for 18–24 hours increased chloride transport to 36% of normal. Conclusion These data confirm that R117H is a residual function CFTR mutation that causes a predominant defect in channel gating activity that is potentiated by ivacaftor. Based on these in vitro data, ivacaftor would be expected to enhance CFTR activity in people with the R117H mutation.

PIP2 and Surface Expression Underlie Apo-Calmodulin Dependent Kv7.2/KCNQ2 Current Potentiation

Calmodulin (CaM) availability regulates neuronal excitability by controlling the functional density of the potassium voltage-dependent M-current, but the basis of this fundamental process is not well understood. We have examined the consequences of varying CaM levels on the main constituents of the M-current, which are Kv7.2 and Kv7.3 subunits. We found that CaM increased the functional density of Kv7.2 channels but not that of Kv7.2/3 or Kv7.3. Similar effects were also observed with a mutant CaM unable to bind Ca2+. In agreement with the proposed role in trafficking, CaM augmented the number of channels at the plasma membrane. To address the involvement of PIP2, its abundance was transiently reduced by activating Danio rerio voltage-sensitive phosphatase (Dr-VSP). CaM-dependent Kv7.2 current potentiation was linked to increased resistance to PIP2 depletion. In contrast, sequestration of endogenous CaM led to a reduction in the functional current density for the three Kv7 channel combinations examined (2, 2/3 and 3). These results suggest that CaM is affecting two mechanisms: one leads to an increase in the number of channels at the plasma membrane, and the second involves a PIP2-dependent increase in opening probability

Regulation of the TRPM3 Channel in Planar Lipid Bilayers

Transient Receptor Potential Melastatin 3 (TRPM3) is a non-selective cation channel that can be activated with nifedipine and pregnenolone sulfate (PS). The role of TRPM3 in nociceptive neurons has been linked to the heat sensation and development of heat hyperalgesia during inflammation. However, the functional characterization of TRPM3 and its specific agonists remain poorly understood. To investigate biophysical and pharmacological properties of TRPM3 we aimed to incorporate the purified channel in planar lipid bilayers. TRPM3 was purified from HEK-293 cells using immunoprecipitation technique. The single channel activity was then examined under various conditions. We investigated TRPM3 activity with different agonists. Application of nifedipine (1-10 µM) resulted in dose-dependent increase of open probability of the channel. These results suggest that nifedipine can alone activate TRPM3. Unlike nifedipine, addition to the bilayers of PS, did not induce the channel openings alone and required co-application of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) or clotrimazole. TRPM3 demonstrated outward rectification upon activation with PS/PI(4,5)P2, but linear current-voltage relationship with PS/clotrimazole. In both conditions, TRPM3 tended to inactivate at higher voltages (100-120 mV) and with increased concentrations of PS. Interestingly, in the absence of any agonists TRPM3 exhibited basal channel activity that sustained for several minutes. Previously TRPM3 was proposed to serve as a temperature sensor. We tested temperature sensitivity of TRPM3 and found that in the framework of lipid bilayers the channel did not exhibit any temperature-induced activation in the absence of agonists. In the presence of nifedipine TRPM3 demonstrated weak temperature dependence with Q10of 1.5 at +100 mV and 1.7 at −100 mV. These results indicate that TRPM3 is unlikely to serve as a temperature sensor alone and may involve some alternative molecular mechanisms for temperature sensation.

Ranolazine Attenuates Hypoxia- and Hydrogen Peroxide-induced Increases in Sodium Channel Late Openings in Ventricular Myocytes

Ranolazine attenuates cardiac arrhythmic activity associated with hypoxia and hydrogen peroxide (H2O2) by inhibition of late sodium current (late INa). The mechanism of ranolazine's action on Na channels was investigated using whole-cell and single-channel recording from guinea pig isolated ventricular myocytes. Hypoxia increased whole-cell late INa from -0.48 ± 0.02 to -3.99 ± 0.07 pA/pF. Ranolazine at 3 and 9 μmol/L reduced the hypoxia-induced late INa by 16% ± 3% and 55% ± 3%, respectively. Hypoxia increased the mean open probability and open time of Na-channel late openings from 0.016 ± 0.001 to 0.064 ± 0.007 milliseconds and from 0.693 ± 0.043 to 1.081 ± 0.098 milliseconds, respectively. Ranolazine at 3 and 9 μmol/L attenuated the hypoxia-induced increase of open probability by 19% ± 7% and 61% ± 1%, and increase of open time by 26% ± 19% and 74 ± 21%, respectively. H2O2 increased the mean open probability and open time of Na-channel late openings from 0.013 ± 0.002 to 0.107 ± 0.015 milliseconds and from 0.689 ± 0.075 to 1.487 ± 0.072 milliseconds, respectively. Ranolazine at 3 and 6 μmol/L reduced the H2O2-induced increase of mean open probability by 60% ± 7% and 95% ± 2%, and the increase of mean open time by 31% ± 21% and 82% ± 8%. In conclusion, the inhibition by ranolazine of hypoxia- and H2O2-stimulated late INa is due to reduction of both the open probability and open time of Na-channel late openings.

Novel roles of raft domain in the molecular mechanism of activation of epithelial Na+ channel at the plasma membrane (892.26)

ENaC expressed in the apical membrane of epithelial cells plays a critical role in homeostasis of fluid content and blood pressure. Na+ transport via ENaC depends on not only the open probability of the channel, but also the number of channel expressed on the apical membrane. ENaC is cleaved by aldosterone‐induced proteases, resulting in an increase in open probability. The lifetime of ENaC at the apical membrane is regulated by ubiquitin ligase (Nedd4‐2). To elucidate roles of raft domain in the regulation of ENaC activation, we quantified the abundance and localization of ENaC at the plasma membrane with/without aldosterone stimulation in renal epithelial cells. Aldosterone stimulated expression of both full‐length and cleaved ENaCs; i.e., the full‐length ENaC was expressed in both cytoplasm and plasma membrane (raft domain), whereas a large portion of the cleaved ENaC was observed at the non‐raft plasma membrane. Depletion of cholesterol from the raft domain diminished expression of cleaved ENaC not only in raft but also non‐raft domain by aldosterone stimulation. Aldosterone also increased expression of Nedd4‐2 and SGK1 in raft domain. Together, these results suggest that the ubiquitination‐mediated regulation of ENaC would occur in raft domain. Our findings demonstrate novel roles of the lipid raft domain on the regulation of ENaC activity/trafficking.

Modification of Cardiac Ryanodine Receptor Gating by a Peptide from the Central Domain of the RyR2

The effect of a domain peptide DPCPVTc from the central region of the RYR2 on ryanodine receptors isolated from rat heart was examined in planar lipid bilayers. At a zero holding potential and at 100 nM cytosolic and 8 mM luminal Ca2+ concentration, DPCPVTc induced concentration-dependent activation of the ryanodine receptor that led up to 20-fold increase of open probability at saturating DPCPVTc concentrations. The effect of the peptide appeared within 30 s after addition to the experimental chamber. At all DPCPVTc concentrations RyR2 channels displayed large variability in open probability, open time and opening frequency. DPCPVTc prolonged RyR2 openings up to 8× and increased RyR2 opening frequency by up to 100%. With increasing DPCPVTc concentration, the fraction of high open probability records increased up to 5×, and their open time increased up to 4×. The closed times did not depend on DPCPVTc concentration either in low- or high-open probability records. The DPCPVTc concentration dependence of all gating parameters had EC50 of 20 µM and a Hill slope of 2. Comparison of the effects of DPCPVTc with those of ATP [1] and cytosolic Ca2+ [2] suggests that activation does not involve luminal feed-through and is not caused by modulation of the cytosolic activation A-site. The data suggest that although “domain unzipping” by DPCPVTc occurs in both modes of RyR activity, it affects RyR gating only when the channel resides in the H-mode of activity.Supported in part by the European Union Contract No. LSHM-CT-2005-018802/CONTICA and by grants APVV-LPP-0441-09, APVV-0628-10 and VEGA 2/0197/11.[1] Tencerova B, Zahradnikova A, Gaburjakova J, Gaburjakova M. J Gen Physiol 140: 93, 2012.[2] Gaburjakova J, Gaburjakova M. J Membr Biol 212: 17, 2006.

Breaking the silence: functional expression of the two-pore-domain potassium channel THIK-2

THIK-2 belongs to the 'silent' channels of the two-pore-domain potassium channel family. It is highly expressed in many nuclei of the brain but has so far resisted all attempts at functional expression. THIK-2 has a highly conserved 19-amino-acid region in its N terminus (residues 6-24 in the rat orthologue) that is missing in the closely related channel THIK-1. After deletion of this region (THIK-2(Δ6-24) mutant), functional expression of the channel was observed in Xenopus oocytes and in mammalian cell lines. The resulting potassium current showed outward rectification under physiological conditions and slight inward rectification with symmetrical high-K(+) solutions and could be inhibited by application of halothane or quinidine. Another THIK-2 mutant, in which the putative retention/retrieval signal RRR at positions 14-16 was replaced by AAA, produced a similar potassium current. Both mutants showed a distinct localisation to the surface membrane when tagged with green fluorescent protein and expressed in a mammalian cell line, whereas wild-type THIK-2 was mainly localised to the endoplasmic reticulum. These findings suggest that deletion of the retention/retrieval signal RRR enabled transport of THIK-2 channels to the surface membrane. Combining the mutation THIK-2(Δ6-24) with a mutation in the pore cavity (rat THIK-2(I158G)) gave rise to a 12-fold increase in current amplitude, most likely due to an increase in open probability. In conclusion, the characteristics of THIK-2 channels can be analysed in heterologous expression systems by using trafficking and/or gating mutants. The possible mechanisms that enable THIK-2 expression at the surface membrane in vivo remain to be determined.

ENaC gating is coupled to the distance between the α‐and βENaC subunits at the base of the thumb and wrist domains

The extracellular domain of ENaC functions as a sensor that modulates channel activity in response to changing extracellular conditions. However, the mechanism by which the extracellular domain influences channel gating is not known. In previous work, we determined that αK477 plays a role in ENaC pH sensing. Based on the ASIC1a crystal structure, we predict that αK477 forms an interface with βV85 and hypothesize that movement at this interface is coupled to channel gating. In the current work, we investigated potential intersubunit interactions at the single channel level. In Xenopus oocytes expressing αK477CβV85CγENaC, treatment with a long cysteine reactive cross‐linking reagent (MTS‐14‐MTS) resulted in an increase in open probability (Po) from 0.4 (± .06) to 0.6 (± .04, p = .04). Because ENaC Po varies widely, we introduced a mutation in the γENaC subunit that prevents endogenous cleavage and causes the channel to favor the closed state (Po = 0.03 ± .06). MTS‐14‐MTS increased the Po of these channels four fold to 0.12 (± .06, p = .02). To test the effect of a short cross‐linking reagent (MTS‐2‐MTS), we performed single channel studies in low extracellular Li+ conditions to cause the channel to favor the open state (Po = 0.67 ± .13). Under these conditions, treatment with MTS‐2‐MTS decreased ENaC Po to 0.2 (± .08, p = .01). The data suggest that movement at the interface between αK477 and βV85 influences channel gating, providing a potential link between extracellular pH sensing, extracellular domain conformational changes, and channel gating.DMC was supported by NIH 2T32HL007121–36 and PMS by HL072256

Selective modulation of different GABAA receptor isoforms by diazepam and etomidate in hippocampal neurons

Diazepam modulation of native γ2-containing GABAA (γGABAA) receptors increases channel conductance by facilitating protein interactions involving the γ2-subunit amphipathic (MA) region, which is found in the cytoplasmic loop between transmembrane domains 3 and 4 (Everitt et al., 2009). However, many drugs, predicted to act on different GABAA receptor subtypes, increase channel conductance leading us to hypothesize that conductance variation in GABAA receptors may be a general property, mediated by protein interactions involving the cytoplasmic MA stretch of amino acids. In this study we have tested this hypothesis by potentiating extrasynaptic GABAA currents with etomidate and examining the ability of peptides mimicking either the γ2- or δ-subunit MA region to affect conductance. In inside-out hippocampal patches from newborn rats the general anesthetic etomidate potentiated GABA currents, producing either an increase in open probability and single-channel conductance or an increase in open probability, as described previously (Seymour et al., 2009). In patches displaying high conductance channels application of a δ(392–422) MA peptide, but not a scrambled version or the equivalent γ2(381–403) MA peptide, reduced the potentiating effects of etomidate, significantly reducing single-channel conductance. In contrast, when GABA currents were potentiated by the γ2-specific drug diazepam the δ MA peptide had no effect. These data reveal that diazepam and etomidate potentiate different extrasynaptic GABAA receptor subtypes but both drugs modulate conductance similarly. One interpretation of the data is that these drugs elicit potentiation through protein interactions and that the MA peptides compete with these interactions to disrupt this process.

Alternative Splicing at C Terminus of CaV1.4 Calcium Channel Modulates Calcium-dependent Inactivation, Activation Potential, and Current Density

The Ca(V)1.4 voltage-gated calcium channel is predominantly expressed in the retina, and mutations to this channel have been associated with human congenital stationary night blindness type-2. The L-type Ca(V)1.4 channel displays distinct properties such as absence of calcium-dependent inactivation (CDI) and slow voltage-dependent inactivation (VDI) due to the presence of an autoinhibitory domain (inhibitor of CDI) in the distal C terminus. We hypothesized that native Ca(V)1.4 is subjected to extensive alternative splicing, much like the other voltage-gated calcium channels, and employed the transcript scanning method to identify alternatively spliced exons within the Ca(V)1.4 transcripts isolated from the human retina. In total, we identified 19 alternative splice variations, of which 16 variations have not been previously reported. Characterization of the C terminus alternatively spliced exons using whole-cell patch clamp electrophysiology revealed a splice variant that exhibits robust CDI. This splice variant arose from the splicing of a novel alternate exon (43*) that can be found in 13.6% of the full-length transcripts screened. Inclusion of exon 43* inserts a stop codon that truncates half the C terminus. The Ca(V)1.4 43* channel exhibited robust CDI, a larger current density, a hyperpolarized shift in activation potential by ∼10 mV, and a slower VDI. Through deletional experiments, we showed that the inhibitor of CDI was responsible for modulating channel activation and VDI, in addition to CDI. Calcium currents in the photoreceptors were observed to exhibit CDI and are more negatively activated as compared with currents elicited from heterologously expressed full-length Ca(V)1.4. Naturally occurring alternative splice variants may in part contribute to the properties of the native Ca(V)1.4 channels.

Cav1.2 calcium channel is glutathionylated during oxidative stress in guinea pig and ischemic human heart

Glutathionylation as a posttranslational modification of proteins is becoming increasingly recognized, but its role in many diseases has not been demonstrated. Oxidative stress and alterations in calcium homeostasis are associated with the development of cardiac hypertrophy. Because the cardiac L-type Ca2+ channel can be persistently activated after exposure to H2O2, the aim of this study was to determine whether alterations in channel function were associated with glutathionylation of the α1C subunit (Cav1.2) channel protein. Immunoblot analysis indicated that Cav1.2 protein is significantly glutathionylated after exposure to H2O2 and glutathione in vitro and after ischemia–reperfusion injury. L-type Ca2+ channel macroscopic current and intracellular calcium were significantly increased in myocytes after exposure to oxidized glutathione and reversed by glutaredoxin. The increase in current correlated with an increase in open probability of the channel assessed as changes in single-channel activity after exposing the human long N-terminal Cav1.2 to H2O2 or oxidized glutathione. We also demonstrate that the Cav1.2 channel is significantly glutathionylated in ischemic human heart. We conclude that oxidative stress is associated with an increase in glutathionylation of the Cav1.2 channel protein. We suggest that the associated constitutive activity contributes to the development of pathology in ischemic heart disease.

Second transmembrane domain modulates epithelial sodium channel gating in response to shear stress

Na(+) absorption and K(+) secretion in the distal segments of the nephron are modulated by the tubular flow rate. Epithelial Na(+) channels (ENaC), composed of α-, β-, and γ-subunits respond to laminar shear stress (LSS) with an increase in open probability. Higher vertebrates express a δ-ENaC subunit that is functionally related to the α-subunit, while sharing only 35% of sequence identity. We investigated the response of δβγ channels to LSS. Both the time course and magnitude of activation of δβγ channels by LSS were remarkably different from those of αβγ channels. ENaC subunits have similar topology, with an extracellular region connected by two transmembrane domains with intracellular N and C termini. To identify the specific domains that are responsible for the differences in the response to flow of αβγ and δβγ channels, we generated a series of α-δ chimeras and site-specific α-subunit mutants and examined parameters of activation by LSS. We found that specific sites in the region encompassing and just preceding the second transmembrane domain were responsible for the differences in the magnitude and time course of channel activation by LSS.

Potential Role of TRP Channels in Cough Hypersensitivity?

Cough is one of the commonest symptoms of medical importance. The mechanism whereby this protective reflex, vital for the integrity of the airway becomes excessively stimulated has recently been elucidated. The tickly sensation which provokes the urge to cough is invested in a series of nococeptors, which become upregulated in pathological states. In both acute and chronic cough, hypersensitivity to noxious stimuli can be objectively demonstrated. Members of a class of ion channels of the Transient Receptor Potential family act as irritant receptors responding to a wide variety of stimuli. The archetypal receptor, TRPV1 is stimulated by heat, acid, and the pungent extract of peppers, capsaicin. TRPV1 is upregulated by inflammatory mediators. Over expression and agonist induced increase in opening probability of the ion channel leads to increased sensitivity in the upper airway. The related TRPA1 is co-localised with TRPV1 and responds to a wide range of environmental stimuli through non-specific binding. It is likely that the combination of these two receptors is responsible for the almost universal complaint from patients with chronic cough of exquisite sensitivity to previously innocuous stimuli. The term, the Cough Hypersensitivity Syndrome has been coined to encapsulate the clinical manifestations of this up-regulation which occurs in the majority of patients presenting to the cough clinic.

Specific Sites within the Ligand-Binding Domain and Ion Channel Linkers Modulate NMDA Receptor Gating

Gating in the NMDA receptor is initiated in the extracellular ligand-binding domain (LBD) and is ultimately propagated via three linkers-S1-M1, M3-S2, and S2-M4-to the ion channel. M3-S2 directly couples LBD movements into channel gating, but the functional and structural contributions of S1-M1 and S2-M4 to the overall gating process are unknown. A scan of substituted cysteines in and around the NMDA receptor S1-M1 and S2-M4 with a bulky cysteine-reactive reagent identified numerous positions that showed potentiation of glutamate-activated as well as leak currents. As indexed by MK801 (dizocilpine hydrogen maleate), an open channel blocker, this potentiation was attributable to an increase in open probability, an interpretation confirmed for a subset of positions with single-channel recordings. The magnitude of this gating effect, acting through S1-M1 or S2-M4, was dependent on the intrinsic gating properties of the NMDA receptors, being more effective in the inherently low open probability GluN2C- than the higher open probability GluN2A-subunit-containing receptors. For the majority of these potentiation positions, we propose that alteration of gating arises from steric destabilization of contact interfaces where close apposition of the contacting partners is necessary for efficient channel closure. Our results therefore indicate that the NMDA receptor S1-M1 and S2-M4 linkers are dynamic during gating and can modulate the overall energetics of this process. Furthermore, the results conceptualize a mechanistic, as well as a possible structural, framework for pharmacologically targeting the linkers through noncompetitive and subunit-specific modes of action.

Direct activation of transient receptor potential V1 by nickel ions

TRPV1 is a member of the transient receptor potential (TRP) family of cation channels. It is expressed in sensory neurons of the dorsal root and trigeminal ganglia as well as in a wide range of non-neuronal tissues. The channel proteins serve as polymodal receptors for various potentially harmful stimuli to prevent tissue damage by mediating unpleasant or painful sensations. Using Ca imaging and voltage-clamp recordings, we found that low millimolar doses of Ni2+ (NiSO4) are able to induce non-specific cation currents in a capsaicin-sensitive population of cultured mouse trigeminal ganglion neurons. In addition, we show that NiSO4 elicits intracellular Ca2+ transients and membrane currents in HEK293 and CHO cells heterologously expressing rat TRPV1. The use of voltage ramps from -100 to +100 mV revealed a strong outward rectification of these currents. Application of NiSO4 to the cytoplasmic face of inside-out membrane patches did not induce any currents. However, delivering NiSO4 to the extracellular face during outside-out recordings, we observed a significant increase in open probability paralleled by a decrease in channel conductance. When combined with other TRPV1 agonists, NiSO4 produces a bimodal effect on TRPV1 activity, depending on the strength and concentration of the second stimulus. Outwardly directed currents induced by low doses of capsaicin and nearly neutral pH values ( approximately pH = 7.0-6.5) were augmented by low doses of NiSO4. In contrast, responses to stronger stimuli were reduced by NiSO4. Moreover, we were able to identify amino acids involved in the effect of NiSO4 on TRPV1.

The unliganded long isoform of estrogen receptor beta stimulates brain ryanodine receptor single channel activity alongside with cytosolic Ca2+

Ca2+ release from intracellular stores mediated by endoplasmic reticulum membrane ryanodine receptors (RyR) plays a key role in activating and synchronizing downstream Ca2+-dependent mechanisms, in different cells varying from apoptosis to nuclear transcription and development of defensive responses. Recently discovered, atypical “nongenomic” effects mediated by estrogen receptors (ER) include rapid Ca2+ release upon estrogen exposure in conditions implicitly suggesting involvement of RyRs. In the present study, we report various levels of colocalization between RyR type 2 (RyR2) and ER type β (ERβ) in the neuronal cell line HT-22, indicating a possible functional interaction. Electrophysiological analyses revealed a significant increase in single-channel ionic currents generated by mouse brain RyRs after application of the soluble monomer of the long form ERβ (ERβ 1). The effect was due to a strong increase in open probability of RyR higher open channel sublevels at cytosolic [Ca2+] concentrations of 100 nM, suggesting a synergistic action of ERβ1 and Ca2+ in RyR activation, and a potential contribution to Ca2+-induced Ca2+ release rather than to basal intracellular Ca2+ concentration level at rest. This RyR/ERβ interaction has potential effects on cellular physiology, including roles of shorter ERβ isoforms and modulation of the RyR/ERβ complexes by exogenous estrogens.