Inside-out Patch-clamp Recordings Research Articles

Optimization of CFTR gating through the evolution of its extracellular loops.

CFTR chloride channel mutations cause the lethal and incurable disease cystic fibrosis (CF). CFTR is activated by phosphorylation, and phosphorylated channels exhibit "bursting" behavior-"bursts" of openings separated by short "flickery" closures and flanked by long "interburst" closures-driven by ATP binding/hydrolysis at two nucleotide-binding domains. The human channel (hCFTR) and the distant zebrafish ortholog (zCFTR) display differences both in their gating properties and structures. In phosphorylated ATP-bound hCFTR, the hR117 side chain, conserved across evolution, forms an H-bond that stabilizes the open state. Lack of that bond in the hR117H mutant causes CF. In the phosphorylated ATP-bound zCFTR structure that H-bond is not observable. Here, we show that the zR118H mutation does not affect the function of zCFTR. Instead, we identify an H-bond between the zS109 and zS120 side chains of phosphorylated ATP-bound, but not of unphosphorylated apo-, zCFTR. We investigate the role of that interaction using thermodynamic mutant cycles built on gating parameters determined in inside-out patch clamp recordings. We find that zS109 indeed forms an H-bond with zN120 in the flickery closed state, but not in the open or interburst closed states. Although in hCFTR an isoleucine (hI119) replaces the asparagine, mutation hS108A produces a strong hR117H-like phenotype. Since the effects of the latter two mutations are not additive, we conclude that in hCFTR these two positions interact, and the hS108-hR117 and hR117-hE1124 H-bonds cooperate to stabilize the open state. These findings highlight an example of how the gating mechanism was optimized during CFTR molecular evolution.

Exploring functional conservation of the cooling agent binding site in the transient receptor potential melastatin subfamily.

Transient Receptor Potential (TRP) cation channels act as cellular sensors of a wide variety of stimuli and play a role in many biological processes, making them attractive pharmacological targets. However, the lack of specific agonists or antagonists for many subfamily members has impeded studies of both their biophysical properties and their broader biological functions. The architecture of the TRP channel transmembrane domain, which contains both the ion conducting pore and several ligand binding sites, is conserved across the family, and previous studies transferring ligand sensitivity among TRP channels revealed conserved channel gating mechanisms within the Vanilloid subfamily. Sequence-based and structure-based alignments have shown conservation within other TRPM family members of key residues mediating TRPM8 sensitivity to cooling agents and antagonists, even though no other TRPM channels have been previously described to be sensitive to cooling agents. We have been particularly interested in TRPM4 because few pharmacological agents are known to modulate the activity of this channel and it is expressed in many tissues, including cardiac, immune, and cancer cells, and mutations have been causally linked to heritable Familial Heart Block. We have explored the sensitivity of TRPM4 to TRPM8 ligands using inside-out patch clamp recordings and found that cooling agents can modulate TRPM4 activity in response to intracellular Ca2+ and that this sensitivity likely arises from the equivalent binding pocket to TRPM8. This structural and functional conservation may facilitate the use of the rich pharmacology of TRPM8 to guide identification or development of TRPM4-specific ligands.

Understanding impact of δF508 and G551D CFTR mutations on CFTR/PKA-c interaction.

The CFTR anion channel is an ATP Binding Cassette protein and consists of two pore-forming transmembrane domains, two cytosolic nucleotide binding domains that bind and hydrolyze ATP to drive gating, and a cytosolic regulatory (R) domain. The catalytic subunit of protein kinase A (PKA) binds to, and phosphorylates, the R domain, causing reversible and irreversible channel activation, respectively. CFTR chloride channel mutations cause the lethal and incurable disease cystic fibrosis (CF). The most common CF mutation, ΔF508, is known to impair CFTR protein processing and channel gating, while mutation G551D disrupts ATP-dependent channel gating. In addition, ΔF508 and G551D CFTR were also shown to be defective in PKA-dependent activation. As the stimulating effect of ivacaftor, the only FDA-approved CFTR potentiator drug, depends on CFTR phosphorylation, understanding the reason for the impaired CFTR-PKA interaction in the mutants is of great significance. The reduced rate of phosphorylation of G551D or ΔF508 channels might be a consequence of impaired PKA binding to CFTR. In the presence of ATP the contributions of PKA binding vs. phosphorylation to overall channel activation are difficult to deconvolve. Therefore, we tested reversible activation by PKA of unphosphorylated and phosphorylated mutant channels gating in P-ATP, an ATP analog that supports CFTR gating but cannot be used for phosphotransfer by PKA. Using macroscopic inside-out patch clamp recordings we compared the rates and fractional amplitudes of reversible stimulation by PKA for the mutants and wild-type CFTR. Our data show that channel activation by reversible binding of PKA is preserved in the mutant CFTR channels. Additional experiments are underway to decipher the molecular mechanism for slowed activation of the mutants.

Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond.

The phosphorylation-activated anion channel cystic fibrosis transmembrane conductance regulator (CFTR) is gated by an ATP hydrolysis cycle at its two cytosolic nucleotide-binding domains, and is essential for epithelial salt-water transport. A large number of CFTR mutations cause cystic fibrosis. Since recent breakthrough in targeted pharmacotherapy, CFTR mutants with impaired gating are candidates for stimulation by potentiator drugs. Thus, understanding the molecular pathology of individual mutations has become important. The relatively common R117H mutation affects an extracellular loop, but nevertheless causes a strong gating defect. Here, we identify a hydrogen bond between the side chain of arginine 117 and the backbone carbonyl group of glutamate 1124 in the cryo-electronmicroscopic structure of phosphorylated, ATP-bound CFTR. We address the functional relevance of that interaction for CFTR gating using macroscopic and microscopic inside-out patch-clamp recordings. Employing thermodynamic double-mutant cycles, we systematically track gating-state-dependent changes in the strength of the R117-E1124 interaction. We find that the H-bond is formed only in the open state, but neither in the short-lived 'flickery' nor in the long-lived 'interburst' closed state. Loss of this H-bond explains the strong gating phenotype of the R117H mutant, including robustly shortened burst durations and strongly reduced intraburst open probability. The findings may help targeted potentiator design.

Dual Effects of Phosphatidylinositol on TRPV1 Regulation Involve Functionally Distinct Binding Sites

Regulation of the heat- and capsaicin-activated Transient Receptor Potential Vanilloid 1 (TRPV1) channel by phosphoinositides is complex and controversial. While phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a well-established positive regulator, in the TRPV1 cryoEM structure, an endogenous phosphoinositide was detected in the vanilloid binding site and proposed to act as a competitive vanilloid antagonist. How do phosphoinositides act as positive and negative modulators of TRPV1? To answer this, we hypothesized that the antagonist phosphoinositide that occupies the vanilloid binding site is phosphatidylinositol (PtdIns). Excised inside-out patch clamp recordings confirmed that PtdIns, but not PtdIns(4)P, decreased capsaicin-induced TRPV1 currents in the presence of PtdIns(4,5)P2. This negative effect depended on the concentrations of both capsaicin and PtdIns, supporting the competitive binding of the two compounds to the overlapping sites. Surprisingly, in the absence of PtdIns(4,5)P2, PtdIns partially stimulated TRPV1 currents presumably by binding to the activating site when the vanilloid site was occupied by saturating levels of capsaicin. Overall, we showed that PtdIns has dual effects on TRPV1 currents by acting as a positive or negative modulator. These findings are not compatible with a model in which phosphoinositides act through a single binding site. Using a combination of computational and experimental approaches, we propose that phosphoinositides regulate TRPV1 via functionally distinct binding sites.

Mechanisms of Action of the KCa2-Negative Modulator AP30663, a Novel Compound in Development for Treatment of Atrial Fibrillation in Man.

AimsSmall conductance Ca2+-activated K+ channels (SK channels, KCa2) are a new target for treatment of atrial fibrillation (AF). AP30663 is a small molecule inhibitor of KCa2 channels that is currently in clinical development for treatment of AF. The aim of this study is to present the electrophysiological profile and mechanism of action of AP30663 and its efficacy in prolonging atrial refractoriness in rodents, and by bioinformatic analysis investigate if genetic variants in KCNN2 or KCNN3 influence the expression level of these in human heart tissue.Methods and ResultsWhole-cell and inside-out patch-clamp recordings of heterologously expressed KCa2 channels revealed that AP30663 inhibits KCa2 channels with minor effects on other relevant cardiac ion channels. AP30663 modulates the KCa2.3 channel by right-shifting the Ca2+-activation curve. In isolated guinea pig hearts AP30663 significantly prolonged the atrial effective refractory period (AERP) with minor effects on the QT-interval corrected for heart rate. Similarly, in anaesthetized rats 5 and 10 mg/kg of AP30663 changed the AERP to 130.7±5.4% and 189.9±18.6 of baseline values. The expression quantitative trait loci analyses revealed that the genome wide association studies for AF SNP rs13376333 in KCNN3 is associated with increased mRNA expression of KCNN3 in human atrial appendage tissue.ConclusionsAP30663 is a novel negative allosteric modulator of KCa2 channels that concentration-dependently prolonged rodent atrial refractoriness with minor effects on the QT-interval. Moreover, AF associated SNPs in KCNN3 influence KCNN3 mRNA expression in human atrial tissue. These properties support continued development of AP30663 for treatment of AF in man.

Dynamic Regulation of Bicarbonate Permeability through CFTR Channel by WNK1

Increase in relative HCO3− permeability (PHCO3/PCl) of cystic fibrosis transmembrane conductance regulator (CFTR) channel by[Cl−]i-sensitive kinases is essential for pancreatic HCO3− secretion. Defects in pancreatic HCO3− secretion caused by CFTR mutations are associated with cystic fibrosis and pancreatitis. However, the molecular mechanisms by which [Cl−]i-sensitive kinases regulate CFTR anion selectivity are unknown. We examined the mechanisms of CFTR PHCO3/PCl regulation by [Cl−]i-sensitive kinases using the electrophysiological and molecular approach, including whole-cell, outside-out, and inside-out patch clamp recordings and molecular dissection of WNK1 and CFTR proteins. Moreover, we analyzed the effects of pancreatitis-causing CFTR mutations on the WNK1-mediated regulation of CFTR. Although SPAK and/or OSR, which constitute a [Cl−]i-sensitive kinase cascade, wasn't expressed, the expression of WNK1 alone was sufficient to increase the CFTR PHCO3/PCl in patch clamp recordings. In molecular dissection of the WNK1 protein, the WNK1 kinase domain contributes CFTR PHCO3/PCl regulation by direct association with CFTR, while the surrounding N-terminal regions regulate the [Cl−]i-sensitivity of WNK1. Therefore, the inclusion of WNK1 N-terminal recombinant proteins in excised patch clamp recordings increased the CFTR PHCO3/PCl in a dose-dependent manner. The pancreatitis-causing R74Q and R75Q mutations in elbow helix 1 of CFTR hampered WNK1-CFTR physical associations and reduced WNK1-mediated CFTR PHCO3/PCl regulation. The CFTR PHCO3/PCl is regulated by [Cl−]i and a WNK1-dependent mechanism. In low [Cl−]i, physical associations between the N-terminal region of WNK1 and CFTR near p.R75 increase PHCO3/PCl. Defects in this process underlie the pathogenesis of some CFTR-related disorders.

P699Mechanisms of action of the small conductance Ca2+-activated K+-channel modulator AP30663, a novel compound being developed for treatment of atrial fibrillation in man

Abstract Background Small conductance Ca2+-activated K+-channels (SK-channels) are a promising new atrial selective target for treatment of atrial fibrillation (AF). AP30663 is a small molecule inhibitor of SK-channels that is effective in converting vernakalant-resistant AF in tachy-paced pigs. Detailed understanding of the molecular mechanism of AP30663 is important for the development of SK channel inhibition for use in man. Purpose To establish the electrophysiological profile, mechanism of action and efficacy in prolonging atrial refractoriness ex vivo of AP30663. Methods AP30663 potency and mechanism of action were established by whole cell and inside-out patch clamp recordings of expressed SK channels. The ion channel selectivity profile of AP30663 was investigated on heterologous expressed channels. Effects of AP30663 or vehicle (DMSO) on atrial refractoriness (AERP) and ventricular repolarization (QTcB) were investigated on isolated perfused guinea pig hearts. Results AP30663 was found to be a selective negative allosteric modulator of SK channels (IC50=0.77±x0.13 μM) with no or minor effects on a panel of other cardiac ion channels, including hERG/KV11.1, (IKr), KV7.1/KCNE1 (IKs), KV4.3/KChiP2 (Ito), Kir2.1 (IK1), Kir3.1/Kir3.4 (IKACh), KV1.5 (IKur), NaV1.5 (INa) and CaV1.2 (ICa). AP30663 inhibited the SK-channel by right-shifting the calcium activation curve of the SK-channel (the EC50 of Ca2+ increased from 0.43±0.02 μM (control, n=6) to 1.37±0.05 μM (in the presence of 7μM AP30663, n=6). In isolated guinea pig hearts, administration of vehicle had no effect on AERP or QTcB. AP30663 significantly prolonged the AERP in 3 μM (to 131±6% of baseline) and 10 μM (to 165±3% of baseline) without any effects on the QTcB. Conclusion AP30663 is a selective negative allosteric modulator of SK channels, acting by means of shifting the calcium dependence of SK-channel activation. AP30663 prolonged atrial refractoriness without affecting the QT-interval in isolated perfused heart preparations. These properties support continued development of AP30663 for treatment of AF in man. Acknowledgement/Funding Innovation Fund Denmark, Wellcome Trust

Regulation of CFTR Bicarbonate Channel Activity by WNK1: Implications for Pancreatitis and CFTR-Related Disorders.

Backgraoud & AimsAberrant epithelial bicarbonate (HCO3−) secretion caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with several diseases including cystic fibrosis and pancreatitis. Dynamically regulated ion channel activity and anion selectivity of CFTR by kinases sensitive to intracellular chloride concentration ([Cl−]i) play an important role in epithelial HCO3− secretion. However, the molecular mechanisms of how [Cl−]i-dependent mechanisms regulate CFTR are unknown.MethodsWe examined the mechanisms of the CFTR HCO3− channel regulation by [Cl−]i-sensitive kinases using an integrated electrophysiological, molecular, and computational approach including whole-cell, outside-out, and inside-out patch clamp recordings and molecular dissection of WNK1 and CFTR proteins. In addition, we analyzed the effects of pancreatitis-causing CFTR mutations on the WNK1-mediated regulation of CFTR.ResultsAmong the WNK1, SPAK, and OSR1 kinases that constitute a [Cl−]i-sensitive kinase cascade, the expression of WNK1 alone was sufficient to increase the CFTR bicarbonate permeability (PHCO3/PCl) and conductance (GHCO3) in patch clamp recordings. Molecular dissection of the WNK1 domains revealed that the WNK1 kinase domain is responsible for CFTR PHCO3/PCl regulation by direct association with CFTR, while the surrounding N-terminal regions mediate the [Cl−]i-sensitivity of WNK1. Furthermore, the pancreatitis-causing R74Q and R75Q mutations in the elbow helix 1 of CFTR hampered WNK1-CFTR physical associations and reduced WNK1-mediated CFTR PHCO3/PCl regulation.ConclusionThe CFTR HCO3− channel activity is regulated by [Cl−]i and a WNK1-dependent mechanism. Our results provide new insights into the regulation of the ion selectivity of CFTR and the pathogenesis of CFTR-related disorders.

24S-hydroxycholesterol alters activity of large-conductance Ca2+-dependent K+ (slo1 BK) channel through intercalation into plasma membrane

Oxysterols, oxidization products of cholesterol, are regarded as bioactive lipids affecting various physiological functions. However, little is known of their effects on ion channels. Using inside-out patch clamp recording, we found that naturally occurring side-chain oxidized oxysterols, 20S‑hydroxycholesterol, 22R‑hydroxycholesterol, 24S‑hydroxycholestero, 25‑hydroxycholesterol, and 27‑hydroxycholesterol, induced current reduction of large-conductance Ca2+- and voltage-activated K+ (slo1 BK) channels heterologously expressed in HEK293T cells. In contrast with side-chain oxidized oxysterols, naturally occurring ring oxidized ones, 7α‑hydroxycholesterol and 7‑ketocholesterol were without effect. By using 24S‑hydroxycholesterol (24S‑HC), the major brain oxysterol, we explored the inhibition mechanism. 24S‑HC inhibited Slo1 BK channels with an IC50 of ~2 μM, and decreased macroscopic current by ~60%. This marked current decrease was accompanied by a rightward shift in the conductance-voltage relationship and a slowed activation kinetics, with the deactivation kinetics unaltered. Furthermore, the membrane sterol scavenger γ‑cyclodextrin was found to rescue slo1 BK channels from the inhibition, implicating that 24S-HC may be intercalated into the plasma membrane to affect the channel. These findings unveil a novel physiological importance of oxysterols from a new angle that involves ion channel regulation.

Functional characterization of activating mutations in the sulfonylurea receptor 1 (ABCC8) causing neonatal diabetes mellitus in Asian Indian children.

Gain-of-function of ATP-sensitive K+ (KATP ) channels because of mutations in the genes encoding SUR1 (ABCC8) or Kir6.2 (KCNJ11) is a major cause of neonatal diabetes mellitus (NDM). Our aim is to determine molecular defects in KATP channels caused by ABCC8 mutations in Asian Indian children with NDM by in vitro functional studies. Wild-type (WT; NM_000352.4) or mutant sulfonylurea receptor 1 (SUR1) and Kir6.2 were co-expressed in COSm6 cells. Biogenesis efficiency and surface expression of mutant channels were assessed by immunoblotting and immunostaining. The response of mutant channels to cytoplasmic ATP and ADP was assessed by inside-out patch-clamp recordings. The response of mutant channels to known KATP inhibitors in intact cells were determined by 86 Rb efflux assays. Five SUR1 missense mutations, D212Y, P254S, R653Q, R992C, and Q1224H, were studied and showed increased activity in MgATP/MgADP. Two of the mutants, D212Y and P254S, also showed reduced response to ATP4- inhibition, as well as markedly reduced surface expression. Moreover, all five mutants were inhibited by the KATP channel inhibitors glibenclamide and carbamazepine. The study shows the mechanisms by which five SUR1 mutations identified in Asian Indian NDM patients affect KATP channel function to cause the disease. The reduced ATP4- sensitivity caused by the D212Y and P254S mutations in the L0 of SUR1 provides novel insight into the role of L0 in channel inhibition by ATP. The results also explain why sulfonylurea therapy is effective in two patients and inform how it should be effective for the other three patients.

Selectivity and Characterization of the Permeant Ion Effect in the Rapidtransitions on the Pore of TRPV1 Channel

The transient receptor potential vanilloid 1 (TRPV1) channel can be activated by stimuli such as capsaicin, pH <5.4, heat and voltage. Its structure is similar to voltage-dependent potassium channels, but in contrast the mechanism voltage detection has remained unknown.TRPV1 is classified as a non-selective cation channel with more permeability to divalent ions as calcium. We characterized the selectivity of TRPV1 through inside-out patch clamp recordings in cells HEK293. We find a selectivity sequence for monovalent ions: Cs+ ≈ NH4+ > Rb+ ≈ K+ > Na+ > Li+. Additionally, we find that, in single-channels recordings, there are rapid fluctuations on the current that occur during the open state of the channel. These fluctuations are observed as an asymmetric tail in the all-points current amplitude histograms. This asymmetry increases at positive voltages and varies depending on the permeant ion. These fluctuations can be explained by rapid transition between the open state and several closed states. The lifetime of the open state is related to the type of ion that occupies the channel pore. Amplitude histograms are analyzed with through simulation of histograms resulting from several models considering one open state and two or more closed states with appropriate transition rate constants. These experiments indicate that the selectivity filter undergoes rapid transitions that might be related to gating and might reflect the movement of a selectivity filter gate. seems to occur in the selectivity filter. Supported by grants DGAPA-PAPIIT: IN203318 and CONACYT: 252644.

TMEM16F/ANO6, a Ca2+-activated anion channel, is negatively regulated by the actin cytoskeleton and intracellular MgATP

Anoctamin 6 (ANO6/TMEM16F) is a recently identified membrane protein that has both phospholipid scramblase activity and anion channel function activated by relatively high [Ca2+]i. In addition to the low sensitivity to Ca2+, the activation of ANO6 Cl− conductance is very slow (>3–5 min to reach peak level at 10 μM [Ca2+]i), with subsequent inactivation. In a whole-cell patch clamp recording of ANO6 current (IANO6,w-c), disruption of the actin cytoskeleton with cytochalasin-D (cytoD) significantly accelerated the activation kinetics, while actin filament-stabilizing agents (phalloidin and jasplakinolide) commonly inhibited IANO6,w-c. Inside-out patch clamp recording of ANO6 (IANO6,i-o) showed immediate activation by raising [Ca2+]i. We also found that intracellular ATP (3 mM MgATP in pipette solution) decelerated the activation of IANO6,w-c, and also prevented the inactivation of IANO6,w-c. However, the addition of cytoD still accelerated both activation and inactivation of IANO6,w-c. We conclude that the actin cytoskeleton and intracellular ATP play major roles in the Ca2+-dependent activation and inactivation of IANO6,w-c, respectively.

Identification of critical amino acids in the proximal C-terminal of TREK-2 K+ channel for activation by acidic pHi and ATP-dependent inhibition.

TWIK-related two-pore domain K+ channels (TREKs) are regulated by intracellular pH (pHi) and Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Previously, Glu306 in proximal C-terminal (pCt) of mouse TREK-1 was identified as the pHi-sensing residue. The direction of PI(4,5)P2 sensitivity is controversial, and we have recently shown that TREKs are inhibited by intracellular ATP via endogenous PI(4,5)P2 formation. Here we investigate the anionic and cationic residues of pCt for the pHi and ATP-sensitivity in human TREK-2 (hTREK-2). In inside-out patch clamp recordings (ITREK-2,i-o), acidic pHi-induced activation was absent in E332A and was partly attenuated in E335A. Neutralization of cationic Lys (K330A) also eliminated the acidic pHi sensitivity of ITREK-2,i-o. Unlike the inhibition of wild-type (WT) ITREK-2,i-o by intracellular ATP, neither E332A nor K330A was sensitive to ATP. Nevertheless, exogenous PI(4,5)P2 (10μM) abolished ITREK-2 i-o in all the above mutants as well as in WT, indicating unspecific inhibition by exogenous PI(4,5)P2. In whole-cell recordings of TREK-2 (ITREK-2,w-c), K330A and E332A showed higher or fully active basal activity, showing attenuated or insignificant activation by 2-APB, arachidonic acid, or acidic pHe 6.9. ITREK-1,w-c of WT is largely suppressed by pHe 6.9, and the inhibition is slightly attenuated in K312A and E315A. The results show concerted roles of the oppositely charged Lys and Glu in pCt for the ATP-dependent low basal activity and pHi sensitivity.

A genetic variant of the sperm-specific SLO3 K+ channel has altered pH and Ca2+ sensitivities

To fertilize an oocyte, sperm must first undergo capacitation in which the sperm plasma membrane becomes hyperpolarized via activation of potassium (K+) channels and resultant K+ efflux. Sperm-specific SLO3 K+ channels are responsible for these membrane potential changes critical for fertilization in mouse sperm, and they are only sensitive to pH i However, in human sperm, the major K+ conductance is both Ca2+- and pH i -sensitive. It has been debated whether Ca2+-sensitive SLO1 channels substitute for human SLO3 (hSLO3) in human sperm or whether human SLO3 channels have acquired Ca2+ sensitivity. Here we show that hSLO3 is rapidly evolving and reveal a natural structural variant with enhanced apparent Ca2+ and pH sensitivities. This variant allele (C382R) alters an amino acid side chain at a principal interface between the intramembrane-gated pore and the cytoplasmic gating ring of the channel. Because the gating ring contains sensors to intracellular factors such as pH and Ca2+, the effectiveness of transduction between the gating ring and the pore domain appears to be enhanced. Our results suggest that sperm-specific genes can evolve rapidly and that natural genetic variation may have led to a SLO3 variant that differs from wild type in both pH and intracellular Ca2+ sensitivities. Whether this physiological variation confers differences in fertility among males remains to be established.

Existence of ATP sensitive potassium currents on human periodontal ligament cells

ObjectivePotassium channels of the ATP-sensitive family (KATP channel) are inhibited by increase in intracellular ATP. Electrophysiological studies have demonstrated that the kinetics and pharmacological properties of KATP channels vary among different tissues, suggesting structurally and functionally distinct types. There are studies showing human periodontal ligament (PDL) cells respond to mechanical stress by increasing ATP release, which participates in bone resorption or bone homeostasis. So, in this study we investigated the existence of KATP channel subunit and their single channel properties in human periodontal ligaments. Materials & methodThe human PDL cells were isolated from healthy erupted third molar. For patch-clamp experiments, human PDL fibroblasts were seeded on 3.5cm plastic dishes. The inside-out patch clamp recordings were performed under voltage clamp mode. Reverse transcriptase polymerase chain reaction (RT-PCR) was conducted to identify the channel subunits. All pair-wise comparisons were performed by Paired t-test. A P value <0.05 was considered significant. ResultsWe observed mRNA transcripts for Kir6.1, Kir6.2 and Sur2B subuits in the human PDL cells. In inside-out patch mode, the single channel conductance was 163pS at symmetrical K+ concentration of 140mM and inward rectification was seen in ATP-free bath solution. The reversal potential of the currents was found to be 0mV at symmetrical concentration (140mM) of K+ in bath solution. The single channel currents were almost blocked by adding 5mM ATP in the bath solution. However, the currents were not blocked by 100μM glibenclamide, a subunit specific KATP channel blocker. ConclusionsThese results indicate that human PDL cells express KATP channels subunit including Sur2B and Kir6.1 and Kir6.2 which are sensitive to ATP but insensitive to glibenclamide.

Extracellular protons enable activation of the calcium-dependent chloride channel TMEM16A.

The calcium-activated chloride channel TMEM16A provides a pathway for chloride ion movements that are key in preventing polyspermy, allowing fluid secretion, controlling blood pressure, and enabling gastrointestinal activity. TMEM16A is opened by voltage-dependent calcium binding and regulated by permeant anions and intracellular protons. Here we show that a low proton concentration reduces TMEM16A activity while maximum activation is obtained when the external proton concentration is high. In addition, protonation conditions determine the open probability of TMEM16A without changing its calcium sensitivity. External glutamic acid 623 (E623) is key for TMEM16A's ability to respond to external protons. At physiological pH, E623 is un-protonated and TMEM16A is activated when intracellular calcium increases; however, under acidic conditions E623 is partially protonated and works synergistically with intracellular calcium to activate the channel. These findings are critical for understanding physiological and pathological processes that involve changes in pH and chloride flux via TMEM16A. Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore-forming subunit of a Ca2+ -dependent Cl- channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca2+ . Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H+ ]o ) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch-clamp recordings. We found that increasing the [H+ ]o from 10-10 to 10-5.5 m caused a progressive increase in the chloride current (ICl ) that is described by titration of a protonatable site with pK=7.3. Protons regulate TMEM16A in a voltage-independent manner, regardless of channel state (open or closed), and without altering its apparent Ca2+ sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca2+ ]i . To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the ICl was nearly insensitive to proton concentrations between 10-5.5 and 10-9.0 m. Additionally, ICl of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10-5.5 m, but 10-9.0 m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca2+ ]i .

TRPM4 non-selective cation channels influence action potentials in rabbit Purkinje fibres.

The transient receptor potential melastatin 4 (TRPM4) inhibitor 9-phenanthrol reduces action potential duration in rabbit Purkinje fibres but not in ventricle. TRPM4-like single channel activity is observed in isolated rabbit Purkinje cells but not in ventricular cells. The TRPM4-like current develops during the notch and early repolarization phases of the action potential in Purkinje cells. Transient receptor potential melastatin 4 (TRPM4) Ca(2+)-activated non-selective cation channel activity has been recorded in cardiomyocytes and sinus node cells from mammals. In addition, TRPM4 gene mutations are associated with human diseases of cardiac conduction, suggesting that TRPM4 plays a role in this aspect of cardiac function. Here we evaluate the TRPM4 contribution to cardiac electrophysiology of Purkinje fibres. Ventricular strips with Purkinje fibres were isolated from rabbit hearts. Intracellular microelectrodes recorded Purkinje fibre activity and the TRPM4 inhibitor 9-phenanthrol was applied to unmask potential TRPM4 contributions to the action potential. 9-Phenanthrol reduced action potential duration measured at the point of 50 and 90% repolarization with an EC50 of 32.8 and 36.1×10(-6) mol l(-1), respectively, but did not modulate ventricular action potentials. Inside-out patch-clamp recordings were used to monitor TRPM4 activity in isolated Purkinje cells. TRPM4-like single channel activity (conductance = 23.8 pS; equal permeability for Na(+) and K(+); sensitivity to voltage, Ca(2+) and 9-phenanthrol) was observed in 43% of patches from Purkinje cells but not from ventricular cells (0/16). Action potential clamp experiments performed in the whole-cell configuration revealed a transient inward 9-phenanthrol-sensitive current (peak density = -0.65 ± 0.15 pA pF(-1); n = 5) during the plateau phases of the Purkinje fibre action potential. These results show that TRPM4 influences action potential characteristics in rabbit Purkinje fibres and thus could modulate cardiac conduction and be involved in triggering arrhythmias.

Species-Dependent Effects of Mustard Oil on TRPM8

Allyl isothiocyanate (AITC; mustard oil) confers the typical pungency to condiments such as wasabi and mustard, and has been widely used as a pharmacological tool to study pain and neurogenic inflammation. The somatosensory effects of AITC have been largely attributed to direct activation of TRPA1 and TRPV1, two excitatory Transient Receptor Potential (TRP) ion channels expressed in nociceptor neurons. Nevertheless, a subset of sensory neurons responds to AITC in a TRPA1- and TRPV1-independent manner. We show that AITC activates TRPA1/TRPV1-deficient sensory neurons through activation of TRPM8, a TRP channel involved in cool/cold sensation. Whole-cell and inside-out patch-clamp recordings revealed that AITC rapidly and reversibly activates heterologously expressed TRPM8 in a membrane-delimited manner. Moreover, we found that, at higher doses, AITC induced channel activation is followed by channel block, a process that is more pronounced in the human TRPM8 than in the mouse orthologue. Analysis of chimeric channels combining human and mouse TRPM8 orthologues cytosolic revealed domains that determine this AITC-induced channel blockade. TRPM8 may contribute to the complex psychophysical effects of AITC.

Effects of aerobic exercise training on large-conductance Ca2+-activated K+ channels in rat cerebral artery smooth muscle cells

Large-conductance Ca(2+)-activated K(+) (BKCa) channels provide a negative feedback that regulates vascular tone in the brain circulation. This study investigated the effects of aerobic exercise on gating properties of BKCa channels in rat cerebral artery. Rats were subjected to moderate-intensity exercise at low (EX-3d/w) and high (EX-5d/w) training volume on a motor-driven treadmill, and compared with age-matched sedentary animals (SED). Inside-out (I/O) patch clamp recording was performed to measure gating properties of the BKCa channel. Aerobic exercise induced a reduction in heart rate and body weight in both training groups. Exercise increased the channel activity, which was more pronounced in EX-5d/w than that in EX-3d/w group. Kinetic analysis revealed that (1) the contribution of short open states was elevated and the duration of both short and long open states were extended by exercising in EX-3d/w; (2) Ex-3d/w had no significant change on conformation of close states; (3) EX-3d/w increased the mean open time without changing mean closed time; (4) EX-5d/w increased both the contribution and duration of long open states; (5) EX-5d/w increased channel mean open time while decreased mean closed time. The results suggest that regular aerobic exercise may enhance BKCa channel activity in cerebral arterial myocytes by changing its biophysical properties, and the electrical remolding induced by exercise may be training volume-dependent.