Single-channel Conductance Research Articles

Single-channel biophysical and pharmacological characterizations of native human large-conductance calcium-activated potassium channels in freshly isolated detrusor smooth muscle cells.

Recent studies have demonstrated the importance of large-conductance Ca(2+)-activated K(+) (BK) channels in detrusor smooth muscle (DSM) function in vitro and in vivo. However, in-depth characterization of human native DSM single BK channels has not yet been provided. Here, we conducted single-channel recordings from excised patches from native human DSM cells. Inside-out and outside-out recordings in high K(+) symmetrical solution (containing 140 mM KCl and ~300 nM free Ca(2+)) showed single-channel conductance of 215-220 pS, half-maximum constant for activation of ~+75 to +80 mV, and low probability of opening (P o) at +20 mV that increased ~10-fold at +40 mV and ~60-fold at +60 mV. Using the inside-out configuration at +30 mV, reduction of intracellular [Ca(2+)] from ~300 nM to Ca(2+)-free decreased the P o by ~85 %, whereas elevation to ~800 nM increased P o by ~50-fold. The BK channel activator NS1619 (10 μM) enhanced the P o by ~10-fold at +30 mV; subsequent application of the selective BK channel inhibitor paxilline (500 nM) blocked the activity. Changes in intracellular [Ca(2+)] or the addition of NS1619 did not significantly alter the current amplitude or single-channel conductance. This is the first report to provide biophysical and pharmacological profiles of native human DSM single BK channels highlighting their importance in regulating human DSM excitability.

Counting of Ion Channels on a Membrane Patch Aided by Patch-Clamp Fluorometry

Direct estimation of the number of channels on a membrane path is important for channel biophysics. It is a classical question and has been addressed elegantly by previous studies. Especially, pioneering researchers took the advantage of fluctuations in membrane conductance caused by ion channels opening and closing. The number of channels, the single-channel current, and the probability of the channel opening can be obtained using the method of non-stationary or stationary fluctuation analysis. Here, we developed a method to count the number of channels by simultaneous electrical recording of channel opening and optical recording of the fluorescence from the green fluorescent protein(GFP) tagged to the channel. Based on the number of channels and the macroscopic current, we first tuned this method using the cyclic-nucleotide gated (CNG) channel, of which the single channel conductance and open probability are well characterized. Then we applied the I-F relationship to the hyperpolarization-activated, cAMP-regulated HCN channel, of which the estimation of single channel conductance has been controversial. We estimated that the number of channels on a piece of membrane patch could read 10,000 to 20,000 and the single channel conductance for mHCN2 channel is about 1.82 pico Siemens.

Low and High pH Gating of Connexin43 Channels

Intracellular pH (pHi) regulation in myocardial tissue is crucial to preserving cardiac function. H+-ions can flow passively at high rates through connexin gap-junctions to neighbouring cells, helping to maintain pHi-uniformity in multicellular tissue. In ventricular myocytes, this form of spatial H+-dissipation is regulated by pHi at the junction.We have studied the response of connexin43, the main ventricular junctional channel-isoform, to high and low pHi. Transjunctional conductance (Gj) was measured by double whole-cell voltage clamp in HeLa or N2A cell-pairs transfected with connexin43. pHi was manipulated with 80mM acetate or 20mM trimethylamine in the superfusate. Acid (<6.7) and alkaline (∼7.5) pHi reversibly reduced (<5min) cell-to-cell Gj by 35±4% and 56±3% respectively (n=4-5), relative to the values at resting pHi (∼7.0). Acute electrical uncoupling at high and low pHi occurred with no change in single channel conductance (2mM halothane; 122±pS, n=250), suggesting a channel-gating mechanism. Cells expressing a truncated C-terminus mutant of connexin43, showed no conductance response to pHi-changes.Using a different approach, we measured apparent junctional H+-permeability (PHapp) through connexin43. One cell of a pair was acid-loaded by photolytically uncaging H+ from an intracellular donor compound (nitrobenzaldehyde), while pHi was confocally imaged in both cells (SNARF-1). Cell-to-cell H+-diffusion was inhibited by β-glycerrhetinic acid (connexin-blocker, 60μM) and was negligible in wild type cell-pairs. In HCO3−-free buffer, presetting pHi to 6.6 or 7.3, reduced PHapp by 81±6% and 76±14%, respectively (n=5-19). PHapp returned to initial values when pHi was allowed to recover. The presence of 5%CO2/22mMHCO3− in the perfusate doubled PHapp.In conclusion, connexin43 gap-junctions exhibit reversible block at both acid and alkaline pHi, possibly by a gating mechanism involving the cytoplasmic C-terminal tail. This suggests a key role for connexin43-channels in coordinating cell-to-cell electrical coupling and spatial pHi regulation during metabolic stress.

Tuning the Single Channel Conductance of Shaker K-Channels

Potassium channels are membrane proteins that allow passage of K+ ions across the hydrophobic core of the membrane. The extremely conserved signature sequence in the residues lining the pore (TTVGYGD) allows discrimination between ions having similar radii (Hegimbotham et al 1994). Despite of this conservation, closely related potassium channels display differences of up to two orders of magnitude in their K+ ion transport rates. Searching for the molecular determinants accounting for such a large difference in transport rates, we made several charge substitutions at the internal entrance of the pore of the low conductance Shaker K-channel. We expressed these variants in Xenopus oocytes and measured their single channel conductance in inside-out membrane patches in 100 and 1000 mM of symmetrical [K+]. The substitution of Pro475 by Asp or Gln increases Shaker's unitary conductance 8 and 4 fold in 100 mM K+, respectively, as shown earlier (Sukhareva, et al., 2003). But at 1000 mM K+ the increases are 4 and 3 fold, respectively. In these high conductance backgrounds (475D or P475Q), we also introduced either negative or positively charged side chains (Asp or Arg) to positions 476 and 479. These positions align with the negatively charged rings that electrostatically control conductance in BK channels. Our results shows that, as in BK and KcsA channels, electrostatics at the inner mouth of the pore plays an important role on increasing the conductance of this Shaker variant, suggesting similar architecture in the pore of low and high conductance K-channels.Financed by Fondecyt 1120819 and the Millennium Initiative (P09-022-F), IDF is a Mecesup Doctoral Fellow.

Functional roles of the amino terminal domain in determining biophysical properties of Cx50 gap junction channels

Communication through gap junction channels is essential for synchronized and coordinated cellular activities. The gap junction channel pore size, its switch control for opening/closing, and the modulations by chemicals can be different depending on the connexin subtypes that compose the channel. Recent structural and functional studies provide compelling evidence that the amino terminal (NT) domains of several connexins line the pore of gap junction channels and play an important role in single channel conductance (γj) and transjunctional voltage-dependent gating (Vj-gating). This article reviews recent studies conducted on a series of mutations/chimeras in the NT domain of connexin50 (Cx50). Functional examination of the gap junction channels formed by these mutants/chimeras shows the net charge number at the NT domain to be an important factor in γj and in Vj-gating. Furthermore, with an increase in the net negative charge at the NT domain, we observed an increase in the γj as well as changes in the parameters of the Boltzmann fit of the normalized steady-state conductance and Vj relationship. Our data are consistent with a structural model where the NT domain of Cx50 lines the gap junction pore and plays an important role in sensing Vj and in the subsequent conformational changes leading to gating, as well as in limiting the rate of ion permeation.

Autosomal recessiveGJA1(Cx43) gene mutations cause oculodentodigital dysplasia by distinct mechanisms

Oculodentodigital dysplasia (ODDD) is mainly an autosomal dominant human disease caused by mutations in the GJA1 gene, which encodes the gap junction protein connexin43 (Cx43). Surprisingly, there have been two autosomal recessive mutations reported that cause ODDD: a single amino acid substitution (R76H) and a premature truncation mutation (R33X). When expressed in either gap junctional intercellular communication (GJIC)-deficient HeLa cells or Cx43-expressing NRK cells, the R76H mutant trafficked to the plasma membrane to form gap junction-like plaques, whereas the R33X mutant remained diffusely localized throughout the cell, including the nucleus. As expected, the R33X mutant failed to form functional channels. In the case of the R76H mutant, dye transfer studies in HeLa cells and electrical conductance analysis in GJIC-deficient N2a cells revealed that this mutant could form functional gap junction channels, albeit with reduced macroscopic and single channel conductance. Alexa 350 dye transfer studies further revealed that the R76H mutant had no detectable negative effect on the function of co-expressed Cx26, Cx32, Cx37 or Cx40, whereas the R33X mutant exhibited significant dominant or trans-dominant effects on Cx43 and Cx40 as manifested by a reduction in wild-type connexin gap junction plaques. Taken together, our results suggest that the trans-dominant effect of R33X together with its complete inability to form a functional channel may explain why patients harboring this autosomal recessive R33X mutant exhibit greater disease burden than patients harboring the R76H mutant.

Managing the complexity of communication: regulation of gap junctions by post-translational modification

Gap junctions are comprised of connexins that form cell-to-cell channels which couple neighboring cells to accommodate the exchange of information. The need for communication does, however, change over time and therefore must be tightly controlled. Although the regulation of connexin protein expression by transcription and translation is of great importance, the trafficking, channel activity and degradation are also under tight control. The function of connexins can be regulated by several post translational modifications, which affect numerous parameters; including number of channels, open probability, single channel conductance or selectivity. The most extensively investigated post translational modifications are phosphorylations, which have been documented in all mammalian connexins. Besides phosphorylations, some connexins are known to be ubiquitinated, SUMOylated, nitrosylated, hydroxylated, acetylated, methylated, and γ-carboxyglutamated. The aim of the present review is to summarize our current knowledge of post translational regulation of the connexin family of proteins.

Novel Mechanism of Mitochondrial Respiration Control through Competition between Hexokinase-2 and Tubulin for VDAC Binding

The voltage dependent anion channel (VDAC) is involved in regulation of metabolite flux across the mitochondrial outer membrane (MOM). Hexokinse II (HK2) is known to bind the MOM where it phosphorylates glucose into glucose-6-phosphate (G6P). High expression of HXK2 is a common phenotype of many cancers, where its concentration can be 200 times of that in noncancerous cells, and is implicated in the Warburg effect. It is believed that VDAC serves as a HXK2 binding site in the MOM. The 15 amino acid N-terminal sequence of HXK2 is responsible for mitochondrial binding and, when conjugated to TAT (TAT-HK2), binds to mitochondria with higher affinity than native HXK2, causing HXK2 detachment. We have previously found that dimeric tubulin reversibly binds and partially blocks VDAC inhibiting metabolite flux across the MOM. Now we show that this binding can be attenuated by TAT-HXK2 peptide as well as by full length HXK2. We have found that TAT-HXK2 and recombinant full length HXK2 inhibit tubulin blockage of VDAC reconstituted into planar lipid bilayers without altering characteristic channel properties such as single channel conductance and selectivity. Binding of HXK2 to VDAC is verified by the generation of high-frequency excess current noise without channel closure. HXK2 bound to VDAC prevents subsequent tubulin binding, but only when added before tubulin, and inhibits tubulin-induced VDAC blockage in a dose dependent manner. Moreover, G6P, which is known to cause HXK2 detachment from the MOM, fully reverses the inhibition of tubulin-VDAC binding. This suggests that HXK2 detachment from VDAC (and hence the MOM) is caused by a HXK2 conformational change upon G6P binding. Thus we propose a novel mechanism of mitochondrial respiration control in cancer cells through the competition between HXK2 and tubulin for VDAC binding.

Towards a Mechanism of TARP Action at AMPA Receptors

Rapid excitatory synaptic transmission is carried out primarily by glutamate-gated AMPA receptors. In the last few years, a number of AMPA receptor auxiliary proteins have been identified which assemble with the AMPAR at synapses to aid in the trafficking and alter the gating of channel. The prototypical auxiliary protein is stargazin, or γ2, which is a member of the transmembrane AMPA receptor regulatory protein (TARP) family. Stargazin slows desensitization and deactivation, increases the frequency of opening to larger conductances and increases the relative efficacy of partial agonists such as kainate. At present, the structural mechanism by which TARPs enhance AMPAR gating is unclear but there are two distinct possibilities. First, TARPs may stabilize the ligand binding domain in more closed conformations, promoting the activation of the channel. And second, TARPs may increase the probability of opening the channel by increasing the efficiency of coupling between changes at the ligand binding domain and channel segments. To distinguish between these alternatives, we used previously characterized ligand binding domain mutations which de-stabilize the closed-bound states of the channel and asked whether stargazin could rescue gating in these mutants. using rapid perfusion in outside-out patches in both desensitizing and non-desensitizing conditions we found that stargazin rescued deficits in gating as measured by the ratio of glutamate to quisqualate responses and single channel conductance, suggesting that TARPs work by stabilizing closed-cleft states of the ligand binding domain. We further tested this hypothesis using FRET to directly measure conformation changes of the ligand binding domain in AMPAR alone or with stargazin. Preliminary FRET experiments are consistent with our electrophysiology data and support the hypothesis that TARPs enhance AMPAR gating by stabilizing closed bound states of the ligand binding domain.

Electrophysiology of Concatameric Pannexin 1 Channels Reveals the Stoichiometry of C-Terminal Autoinhibition

Pannexin 1 (PANX1) is a non-selective ion channel that mediates the uptake of cyanine dyes and release of nucleotides and other metabolites. PANX1 activation and ATP release/dye uptake are regulated by diverse stimuli, including physicochemical factors (e.g., stretch, K+ ions) and signaling by various G protein-coupled and ionotropic receptors. We recently described a unique regulatory mechanism for the release of ATP from apoptotic cells: PANX1 is activated by caspase cleavage of a C-terminal autoinhibitory domain. Current evidence suggests that PANX1 channels are hexameric, and cleavage-resistant subunits can interfere with caspase-dependent activation in a dominant-negative fashion. To explore the subunit stoichiometry required for C-terminal autoinhibition, we engineered concatameric PANX1 constructs with different (from 0 to 6) combinations of truncated and full-length sequences. Formation of concatemers was assessed by the use of single-molecule photobleaching and protein cross-linking. Whole-cell recordings from concatenated PANX1 constructs suggest that at least four intact C-termini are required to inhibit channel activity. In addition, as the number of intact C-termini increased, there was a progressive decrease in single channel conductance, suggesting that individual C-termini may act within the multimeric channel to inhibit channel conductance. These results provide further mechanistic insights into the regulation of PANX1 channels by the C-terminal autoinhibitory domains.

Mapping the Membrane Topography of Segments TH6-TH7 and TH8-TH9 in the Diphtheria Toxin T-Domain Channel

The T-domain of diphtheria toxin senses a low pH to insert into a lipid membrane, form a transmembrane channel, and translocate the attached catalytic domain across the membrane. Previous work has identified three transmembrane segments of T-domain in the open channel state, corresponding to TH5, TH8 and TH9 in the aqueous crystal structure; the amino-terminal region, TH1-TH4, was shown to be translocated across the membrane to the trans side. It was also shown that residues near either end of the TH6-TH7 segment are not translocated, remaining on the cis side of the membrane; the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, so it was concluded that the TH6-TH7 segment resides at the cis interface. Now we have examined this segment further, using the substituted-cysteine accessibility method. We constructed a series of mutant T-domains with a single cysteine residue at positions in TH6-TH7, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific methanethiosulfonate reagents on the channel conductance. For at least 12 of the mutants, the reagent caused a change in the single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. We also compared the reaction rate of reagent added to the cis side vs. that to the trans side in order to estimate the residue's position along the channel axis. This analysis revealed abrupt changes in cis- vs. trans-side accessibility, suggesting that the TH6-TH7 segment forms a constriction that occupies a small portion of the total channel length. The location of this constriction relative to the TH8-TH9 segment was also determined.

Properties of Single HCN2 Channels Expressed in Xenopus Oocytes

Hyperpolarization activated cyclic nucleotide-modulated (HCN) channels mediate rhythmic electrical activity in specialized brain neurons and cardiomyocytes. The channels are non-specific cation channels that are activated by hyperpolarizing voltage. Activation is enhanced by the binding of cAMP to cyclic nucleotide binding domains in each of the four subunits. In mammalians four isoforms of HCN channels have been identified (HCN1-4). The single-channel conductance of HCN channels has been described first in native cardiac channels (DiFrancesco, Nature, 1986). Its value was determined to be only ∼1 pS which is unusually small for a voltage gated cation channel. Surprisingly, in recombinant HCN2 channels a much larger conductance of ∼35 pS was determined (Michels et al., Circulation, 2005). Later Dekker and Yellen (J. Gen. Physiol., 2006) confirmed a small conductance of ∼1.5 pS but observed a pronounced cooperative gating of multiple channels. We expressed HCN2 channels in Xenopus oocytes and studied single channel currents in inside-out patches. Our results confirm a small conductance (∼2 pS) and do not provide any evidence for a cooperative gating between the channels, enabling recording from patches containing one and only one channel. The activating effect of cAMP, applied to the bath solution, is mediated by an increase of the open probability. In conclusion, HCN2 channels expressed in Xenopus oocytes develop an only small single-channel conductance, gate as individual channels, and are activated by cAMP via an increase of the open probability. These results are of importance for modelling single-channel properties from macroscopic currents.

Insights into the Size and Geometry of a Robust Engineered Membrane Protein Nanopore

The maximum utility of protein nanopores in biotechnological applications is generally dictated by the size and internal geometry of the protein. The knowledge of these two parameters is imperative, particularly when extensive protein redesign is employed to produce task-specific proteins.Recently, we used ferric hydroxamate uptake component A (FhuA), a β-barrel bacterial outermembrane protein, as a protein-engineering template to produce an unusually-rigid, open nanopore (FhuA ΔC/Δ4L). This new nanopore maintains its stability under harsh experimental conditions. It was essential to couple comprehensive protein redesign with protein refolding protocols to obtain such a nanopore. With the radical revamp of the FhuA template, it was necessary to shed light on the overall structure of the new FhuA ΔC/Δ4L protein. Thus, we employed water-soluble, flexible poly(ethylene glycols) and dextran polymers to inspect the interior of FhuA ΔC/Δ4L during single-channel recordings.The addition of poly(ethylene glycols) to solution produced alterations in the single-channel conductance, allowing for the calculation of the nanopore diameter. We report that FhuA ΔC/Δ4L features an approximate conical internal geometry with the cis entrance (extracellular) smaller than the trans entrance (periplasmic). This finding is in accord with the asymmetric nature of the crystal structure of the wild-type FhuA protein. Additionally, experiments with impermeable dextran indicated an average internal diameter of ∼2.4 nm, an estimate based on the polymer-induced alteration of the access resistance contribution to the total resistance of the nanopore. The insights into size and geometry of FhuA ΔC/Δ4L deduced from these polymers experiments will aid in future protein engineering of FhuA, opening new engineering avenues for specific tasks in biotechnological applications.This work is funded in part by grants from the US National Science Foundation (DMR-1006332, L.M.) and the National Institutes of Health (R01 GM088403, L.M.).

Histone deacetylase inhibition reduces cardiac connexin43 expression and gap junction communication

Histone deacetylase inhibitors (HDACIs) are being investigated as novel therapies for cancer, inflammation, neurodegeneration, and heart failure. The effects of HDACIs on the functional expression of cardiac gap junctions (GJs) are essentially unknown. The purpose of this study was to determine the effects of trichostatin A (TSA) and vorinostat (VOR) on functional GJ expression in ventricular cardiomyocytes. The effects of HDAC inhibition on connexin43 (Cx43) expression and functional GJ assembly were examined in primary cultured neonatal mouse ventricular myocytes. TSA and VOR reduced Cx43 mRNA, protein expression, and immunolocalized Cx43 GJ plaque area within ventricular myocyte monolayer cultures in a dose-dependent manner. Chromatin immunoprecipitation experiments revealed altered protein interactions with the Cx43 promoter. VOR also altered the phosphorylation state of several key regulatory Cx43 phospho-serine sites. Patch clamp analysis revealed reduced electrical coupling between isolated ventricular myocyte pairs, altered transjunctional voltage-dependent inactivation kinetics, and steady state junctional conductance inactivation and recovery relationships. Single GJ channel conductance was reduced to 54 pS only by maximum inhibitory doses of TSA (≥ 100 nM). These two hydroxamate pan-HDACIs exert multiple levels of regulation on ventricular GJ communication by altering Cx43 expression, GJ area, post-translational modifications (e.g., phosphorylation, acetylation), gating, and channel conductance. Although a 50% downregulation of Cx43 GJ communication alone may not be sufficient to slow ventricular conduction or induce arrhythmias, the development of class-selective HDACIs may help avoid the potential negative cardiovascular effects of pan-HDACI.

Signal processing by T-type calcium channel interactions in the cerebellum.

T-type calcium channels of the Cav3 family are unique among voltage-gated calcium channels due to their low activation voltage, rapid inactivation, and small single channel conductance. These special properties allow Cav3 calcium channels to regulate neuronal processing in the subthreshold voltage range. Here, we review two different subthreshold ion channel interactions involving Cav3 channels and explore the ability of these interactions to expand the functional roles of Cav3 channels. In cerebellar Purkinje cells, Cav3 and intermediate conductance calcium-activated potassium (IKCa) channels form a novel complex which creates a low voltage-activated, transient outward current capable of suppressing temporal summation of excitatory postsynaptic potentials (EPSPs). In large diameter neurons of the deep cerebellar nuclei, Cav3-mediated calcium current (IT) and hyperpolarization-activated cation current (IH) are activated during trains of inhibitory postsynaptic potentials. These currents have distinct, and yet synergistic, roles in the subthreshold domain with IT generating a rebound burst and IH controlling first spike latency and rebound spike precision. However, by shortening the membrane time constant the membrane returns towards resting value at a faster rate, allowing IH to increase the efficacy of IT and increase the range of burst frequencies that can be generated. The net effect of Cav3 channels thus depends on the channels with which they are paired. When expressed in a complex with a KCa channel, Cav3 channels reduce excitability when processing excitatory inputs. If functionally coupled with an HCN channel, the depolarizing effect of Cav3 channels is accentuated, allowing for efficient inversion of inhibitory inputs to generate a rebound burst output. Therefore, signal processing relies not only on the activity of individual subtypes of channels but also on complex interactions between ion channels whether based on a physical complex or by indirect effects on membrane properties.

Measurement of hERG Ion Channel Currents in Lipid Bilayer

hERG channels (human ether-a -go-go related gene, Kv11.1) play an important role conducting potassium ions in the cardiac delayed rectifier current, IKr, during the repolarization phase of the cardiac action potential. We have measured hERG channels in droplet interface bilayers using membrane preparations made from eukaryotic cells expressing hERG. We find single channel conductance and reversal potentials consistent with previously published patch clamp studies as well as the sensitivity of the measured currents to astemizole, a potassium channel blocker, and E-4031, a hERG specific blocker. This sensitivity is dosage dependent, with IC50 values measured, 91 nM and 12.4 nM for astemizole and E-4031, respectively.

Effect of Counter Ions on the Transport Current Across Membranes Containing KAT1 Potassium Channel

Ion transports from one aqueous phase (W1) to another (W2) across bilayer lipid membranes (BLM) containing KAT1 potassium channel were investigated by recording current fluctuations. KAT1 channel is a voltage-gated K(+) channel from Arabidopsis thaliana and has been suggested to have a key role in stomatal opening by osmoregulation in guard cells. Although KAT1 channel is a K(+)-specific transporter, the species of the counter anions significantly affected the level of the single channel current, suggesting that KAT1 also transported these anions. When various potassium salts were used as electrolytes, the single channel conductance decreased with an increase of the ionic radius of the coexisting anions except for F(-). This indicates the existence of selective permeation for anions, but the anion-selectivity is less noticeable than the cation-selectivity.

Tmem16F forms a Ca2+-Activated Cation Channel Required for Lipid Scrambling in Platelets during Blood Coagulation

Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca2+-activated Cl- channels (CaCCs) is linked to Scott Syndrome with deficient Ca2+-dependent lipid scrambling. We generated TMEM16F-knockout mice that exhibit bleeding defects and protection in an arterial thrombosis model associated with platelet deficiency in Ca2+-dependent phosphatidylserine exposure and procoagulant activity, and lack a Ca2+-activated cation current in the platelet precursor megakaryocytes. Heterologous expression of TMEM16F generates a novel Small-conductance Ca2+-Activated Non-selective cation (SCAN) current with sub-picosiemens single channel conductance rather than a CaCC. TMEM16F-SCAN channels permeate both monovalent and divalent cations including Ca2+, and exhibit synergistic gating by Ca2+ and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a novel Ca2+-activated channel permeable to Ca2+ and critical for Ca2+-dependent scramblase activity.

Acidic pH Uncovers Desensitization and Structurally-Distinct Types of Voltage Gating in CNGA1 Channels

Cyclic nucleotide-gated (CNG) channels are members of the superfamily of voltage gated ion channels, but in the presence Na+ and K+ they are gated primarily by cyclic nucleotides (CNs) and only weakly by voltage. Here, we show that when extracellular pH (pHo) is decreased from pH 7.4 to 5 WT CNGA1 channels desensitize, i.e. the current activated by a steady cGMP concentration declines within 5-40 s by 20-80% in a voltage dependent way. Current desensitization is completely reversible upon removal of cGMP and voltage dependency of desensitization is associated to the displacement of 0.3 equivalent electronic charges across the electrical field. A very similar desensitization is observed in several mutant channels, such as E363A and T364A at the usual pH 7.4. At the desensitized state, the I/V relations are outwardly rectifying similarly in the WT CNGA1 channels at pH 5 and mutant channels E363A at pH 7.4. In the presence of symmetrical Rb+ or Cs+, the single channel conductance gsc in WT CNGA1 channels is highly voltage dependent and this voltage dependence is abolished in mutant channels E363A and T364A. CNGA1 channels have structurally-distinct types of voltage sensors, formed by charged and polar residues located in the pore, by the usual S4 voltage sensor and by an additional voltage sensor associated to the voltage dependency of desensitization. Our results not only shed a new light on voltage gating in CNG channels, but uncover the existence of a novel component of phototrasduction: neuronal signalling within the vertebrate retina is highly dependent on the pHo and proton elevation could act as a negative feedback.

The effects of the histone deacetylase inhibitor 4-phenylbutyrate on gap junction conductance and permeability

Longitudinal resistance is a key factor in determining cardiac action potential propagation. Action potential conduction velocity has been shown to be proportional to the square root of longitudinal resistance. A major determinant of longitudinal resistance in myocardium is the gap junction channel, comprised connexin proteins. Within the ventricular myocardium connexin43 (Cx43) is the dominantly expressed connexin. Reduced numbers of gap junction channels will result in an increase in longitudinal resistance creating the possibility of slowed conduction velocity while increased numbers of channels would potentially result in an increase in conduction velocity. We sought to determine if inhibition of histone deacetylase (HDAC) by 4-phenylbutyrate (4-PB), a known inhibitor of HDAC resulted in an increase in junctional conductance and permeability, which is not the result of changes in single channel unitary conductance. These experiments were performed using HEK-293 cells and HeLa cells stably transfected with Cx43. Following treatment with increasing concentrations of 4-PB up-regulation of Cx43 was observed via Western blot analysis. Junctional (gj) conductance and unitary single channel conductance were measured via whole-cell patch clamp. In addition intercellular transfer of lucifer yellow (LY) was determined by fluorescence microscopy. The data in this study indicate that 4-PB is able to enhance functional Cx43 gap junction coupling as indicated by LY dye transfer and multichannel and single channel data along with Western blot analysis. As a corollary, pharmacological agents such as 4-PB have the potential, by increasing intercellular coupling, to reduce the effect of ischemia. It remains to be seen whether drugs like 4-PB will be effective in preventing cardiac maladies.