Single-channel Current Amplitude Research Articles

Genetic Variant in Nicotinic Receptor α4-Subunit Causes Sleep-Related Hyperkinetic Epilepsy via Increased Channel Opening.

We describe genetic and molecular-level functional alterations in the α4β2 neuronal nicotinic acetylcholine receptor (nAChR) from a patient with sleep-related hyperkinetic epilepsy and a family history of epilepsy. Genetic sequencing revealed a heterozygous variant c.851C>G in the CHRNA4 gene encoding the α4 subunit, resulting in the missense mutation p.Ser284Trp. Patch clamp recordings from genetically engineered nAChRs incorporating the α4-Ser284Trp subunit revealed aberrant channel openings in the absence of agonist and markedly prolonged openings in its presence. Measurements of single channel current amplitude distinguished two pentameric stoichiometries of the variant nAChR containing either two or three copies of the α4-Ser284Trp subunit, each exhibiting aberrant spontaneous and prolonged agonist-elicited channel openings. The α4-Ser284 residue is highly conserved and located within the M2 transmembrane α-helix that lines the ion channel. When mapped onto the receptor’s three-dimensional structure, the larger Trp substitution sterically clashes with the M2 α-helix from the neighboring subunit, promoting expansion of the pore and stabilizing the open relative to the closed conformation of the channel. Together, the clinical, genetic, functional, and structural observations demonstrate that α4-Ser284Trp enhances channel opening, predicting increased membrane excitability and a pathogenic seizure phenotype.

VU6036720: The First Potent and Selective In Vitro Inhibitor of Heteromeric Kir4.1/5.1 Inward Rectifier Potassium Channels.

Heteromeric Kir4.1/Kir5.1 (KCNJ10/KCNJ16) inward rectifier potassium (Kir) channels play key roles in the brain and kidney, but pharmacological tools for probing their physiology and therapeutic potential have not been developed. Here, we report the discovery, in a high-throughput screening of 80,475 compounds, of the moderately potent and selective inhibitor VU0493690, which we selected for characterization and chemical optimization. VU0493690 concentration-dependently inhibits Kir4.1/5.1 with an IC50 of 0.96 μM and exhibits at least 10-fold selectivity over Kir4.1 and ten other Kir channels. Multidimensional chemical optimization of VU0493690 led to the development of VU6036720, the most potent (IC50 = 0.24 μM) and selective (>40-fold over Kir4.1) Kir4.1/5.1 inhibitor reported to date. Cell-attached patch single-channel recordings revealed that VU6036720 inhibits Kir4.1/5.1 activity through a reduction of channel open-state probability and single-channel current amplitude. Elevating extracellular potassium ion by 20 mM shifted the IC50 6.8-fold, suggesting that VU6036720 is a pore blocker that binds in the ion-conduction pathway. Mutation of the "rectification controller" asparagine 161 to glutamate (N161E), which is equivalent to small-molecule binding sites in other Kir channels, led to a strong reduction of inhibition by VU6036720. Renal clearance studies in mice failed to show a diuretic response that would be consistent with inhibition of Kir4.1/5.1 in the renal tubule. Drug metabolism and pharmacokinetics profiling revealed that high VU6036720 clearance and plasma protein binding may prevent target engagement in vivo. In conclusion, VU6036720 represents the current state-of-the-art Kir4.1/5.1 inhibitor that should be useful for probing the functions of Kir4.1/5.1 in vitro and ex vivo. SIGNIFICANCE STATEMENT: Heteromeric inward rectifier potassium (Kir) channels comprising Kir4.1 and Kir5.1 subunits play important roles in renal and neural physiology and may represent inhibitory drug targets for hypertension and edema. Herein, we employ high-throughput compound library screening, patch clamp electrophysiology, and medicinal chemistry to develop and characterize the first potent and specific in vitro inhibitor of Kir4.1/5.1, VU6036720, which provides proof-of-concept that drug-like inhibitors of this channel may be developed.

BK Channels Are Activated by Functional Coupling With L-Type Ca2+ Channels in Cricket Myocytes.

Large-conductance calcium (Ca2+)-activated potassium (K+) (BK) channel activation is important for feedback control of Ca2+ influx and cell excitability during spontaneous muscle contraction. To characterize endogenously expressed BK channels and evaluate the functional relevance of Ca2+ sources leading to BK activity, patch-clamp electrophysiology was performed on cricket oviduct myocytes to obtain single-channel recordings. The single-channel conductance of BK channels was 120 pS, with increased activity resulting from membrane depolarization or increased intracellular Ca2+ concentration. Extracellular application of tetraethylammonium (TEA) and iberiotoxin (IbTX) suppressed single-channel current amplitude. These results indicate that BK channels are endogenously expressed in cricket oviduct myocytes. Ca2+ release from internal Ca2+ stores and Ca2+ influx via the plasma membrane, which affect BK activity, were investigated. Extracellular Ca2+ removal nullified BK activity. Administration of ryanodine and caffeine reduced BK activity. Administration of L-type Ca2+ channel activity regulators (Bay K 8644 and nifedipine) increased and decreased BK activity, respectively. Finally, the proximity between the L-type Ca2+ channel and BK was investigated. Administration of Bay K 8644 to the microscopic area within the pipette increased BK activity. However, this increase was not observed at a sustained depolarizing potential. These results show that BK channels are endogenously expressed in cricket oviduct myocytes and that BK activity is regulated by L-type Ca2+ channel activity and Ca2+ release from Ca2+ stores. Together, these results show that functional coupling between L-type Ca2+ and BK channels may underlie the molecular basis of spontaneous rhythmic contraction.

The tarantula toxin GxTx detains K+ channel gating charges in their resting conformation.

Allosteric ligands modulate protein activity by altering the energy landscape of conformational space in ligand-protein complexes. Here we investigate how ligand binding to a K+ channel's voltage sensor allosterically modulates opening of its K+-conductive pore. The tarantula venom peptide guangxitoxin-1E (GxTx) binds to the voltage sensors of the rat voltage-gated K+ (Kv) channel Kv2.1 and acts as a partial inverse agonist. When bound to GxTx, Kv2.1 activates more slowly, deactivates more rapidly, and requires more positive voltage to reach the same K+-conductance as the unbound channel. Further, activation kinetics are more sigmoidal, indicating that multiple conformational changes coupled to opening are modulated. Single-channel current amplitudes reveal that each channel opens to full conductance when GxTx is bound. Inhibition of Kv2.1 channels by GxTx results from decreased open probability due to increased occurrence of long-lived closed states; the time constant of the final pore opening step itself is not impacted by GxTx. When intracellular potential is less than 0 mV, GxTx traps the gating charges on Kv2.1's voltage sensors in their most intracellular position. Gating charges translocate at positive voltages, however, indicating that GxTx stabilizes the most intracellular conformation of the voltage sensors (their resting conformation). Kinetic modeling suggests a modulatory mechanism: GxTx reduces the probability of voltage sensors activating, giving the pore opening step less frequent opportunities to occur. This mechanism results in K+-conductance activation kinetics that are voltage-dependent, even if pore opening (the rate-limiting step) has no inherent voltage dependence. We conclude that GxTx stabilizes voltage sensors in a resting conformation, and inhibits K+ currents by limiting opportunities for the channel pore to open, but has little, if any, direct effect on the microscopic kinetics of pore opening. The impact of GxTx on channel gating suggests that Kv2.1's pore opening step does not involve movement of its voltage sensors.

Alcohol reduces muscle fatigue through atomistic interactions with nicotinic receptors

Alcohol consumption affects many organs and tissues, including skeletal muscle. However, the molecular mechanism of ethanol action on skeletal muscle remains unclear. Here, using molecular dynamics simulations and single channel recordings, we show that ethanol interacts with a negatively charged amino acid within an extracellular region of the neuromuscular nicotinic acetylcholine receptor (nAChR), thereby altering its global conformation and reducing the single channel current amplitude. Charge reversal of the negatively charged amino acid abolishes the nAChR-ethanol interaction. Moreover, using transgenic animals harboring the charge-reversal mutation, ex vivo measurements of muscle force production show that ethanol counters fatigue in wild type but not homozygous αE83K mutant animals. In accord, in vivo studies of motor coordination following ethanol administration reveal an approximately twofold improvement for wild type compared to homozygous mutant animals. Together, the converging results from molecular to animal studies suggest that ethanol counters muscle fatigue through its interaction with neuromuscular nAChRs.

Chloride Single Channel Activity in Freshly‐Isolated Guinea Pig Detrusor Smooth Muscle Cells

Ion channels in detrusor smooth muscle (DSM) control excitability and contractility of the urinary bladder. Several ion channel types have already been detected and characterized in DSM cells, especially voltage‐gated cationic channels. However, little is known about Cl− channels in DSM cells. Specifically, it is unknown what Cl− channel subtypes are expressed in the plasma membrane of DSM cells and how they control DSM cell excitability. Earlier molecular and fluorometric studies suggested the presence of Ca2+‐activated Cl− channels in DSM cells. Our Agilent Human GE 4x44K v2 microarray data analysis on single DSM cell mRNA detected a number of candidates underlying Cl− conductance in DSM cells including plasmalemmal TMEM16A/ANO1, CLCN3, CLCN6, and CLCN7. Thus, detailed biophysical analyses are needed to characterize these Cl− channels. Here, guinea pig DSM cells were freshly isolated by an enzymatic digestion method with papain and collagenase type II, plated at the glass bottom of a recording chamber, and electrophysiologically tested within 6 hours after the isolation. Inside‐out single channel patch clamp recording technique was used to detect electrical activity of Clchannels in the plasma membrane of isolated DSM cells. To separate Cl− from cationic currents, all experiments were done using asymmetrical Cl− and symmetrical Na+ solutions. The bath/intracellular solution contained (in mM): 110 Na‐glutamate, 5 NaCl, 60 mannitol, 10 HEPES, 1 CaCl2, 1 CoCl2, 0.0005 phorbol 12‐myristate 13‐acetate, pH 7.2; the pipette/extracellular contained (in mM): 110 NaCl, 1.5 MgCl2, 2 CaCl2, 60 mannitol, 10 HEPES, 0.001 paxilline, 0.01 nifedipine, pH 7.4. Cl− single channel activity detection frequency varied from zero to 26.7 % within a single DSM cell preparation with an average frequency for detection of 7.4 % (13 out of 175 patches, 8 guinea pigs). Cl− channels displayed unique gating with multiple subconductive states and a fully opened single‐channel conductance in a range from 140 pS to 193 pS with an average value of 163 ± 15 pS (12 patches, 8 guinea pigs, mean ± SD). A linear fitting of single channel current amplitude – membrane potential relationships yielded a reversal potential of − 41.3 ± 5.5 mV (12 patches, 8 guinea pigs, mean ± SD) for this Cl− channel, close to the theoretical ECl of − 65 mV and confirming that this channel is permeable to Cl−. The Cl− channel demonstrated strong voltage‐dependence of activation with virtually no activity below the measured reversal potential and always robust prolonged openings at depolarizing voltages. The channel had similar gating when exposed to the intracellular Ca2+‐free (buffered with 5 mM EGTA in nominal Ca2+) and 1 mM Ca2+ solutions. The Cl− channel was not blocked by 9‐phenanthrol (30 μM), a transient receptor potential melastatin 4 channel inhibitor that also has been reported to block the activity of TMEM16A Ca2+‐activated Cl− channels in vascular smooth muscle cells. In conclusion, our data reveal for the first time the presence of Ca2+‐insensitive voltage‐dependent Cl− channel in guinea pig DSM cells at the single channel level. This channel may be a key regulator of DSM excitability.Support or Funding InformationSupported by NIH R01DK106964 to Georgi V. Petkov.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fluctuation Analysis in Nonstationary Conditions: Single Ca2+ Channel Current in Pyramidal Neurons

Fluctuation analysis is a method that allows measurement of the single-channel current of ion channels even when it is too small to be resolved directly with the patch-clamp technique. This is the case for voltage-gated calcium channels. They are present in all mammalian central neurons, controlling presynaptic release of transmitter, postsynaptic signaling, and synaptic integration. The amplitudes of their single-channel currents in a physiological concentration of extracellular calcium, however, are small and not well determined. But measurement of this quantity is essential for estimating numbers of functional voltage-gated calcium channels in the membrane and the size of channel-associated calcium signaling domains, and for understanding the stochastic nature of calcium signaling. Here, we recorded the voltage-gated calcium channel current in nucleated patches from layer 5 pyramidal neurons in rat neocortex, in physiological external calcium (1–2 mM). The ensemble-averaging of current responses required for conventional fluctuation analysis proved impractical because of the rapid rundown of calcium channel currents. We therefore developed a more robust method, using mean current fitting of individual current responses and band-pass filtering. Furthermore, voltage-ramp stimulation proved useful. We validated the accuracy of the method by analyzing simulated data. At an external calcium concentration of 1 mM, and a membrane potential of −20 mV, we found that the average single-channel current amplitude was ∼0.04 pA, increasing to 0.065 pA at 2 mM external calcium, and 0.12 pA at 5 mM. The relaxation time constant of the fluctuations was in the range 0.2–0.8 ms. The results are relevant to understanding the stochastic properties of dendritic Ca2+ spikes in neocortical layer 5 pyramidal neurons. With the reported method, single-channel current amplitude of native voltage-gated calcium channels can be resolved accurately despite conditions of unstable rundown.

Current-direction/amplitude-dependent single channel gating kinetics of mouse pannexin 1 channel: a new concept for gating kinetics

The detailed single-channel gating kinetics of mouse pannexin 1 (mPanx1) remains unknown, although mPanx1 is reported to be a voltage-activated anion-selective channel. We investigated characteristics of single-channel conductances and opening and closing rates of mPanx1 using patch-clamp techniques. The unitary current of mPanx1 shows outward rectification with single-channel conductances of ~20 pS for inward currents and ~80 pS for outward currents. The channel open time for outward currents (Cl− influx) increases linearly as the amplitude of single channel currents increases, while the open time for inward currents (Cl− efflux) is constant irrespective of changes in the current amplitude, as if the direction and amplitude of the unitary current regulates the open time. This is supported by further observations that replacement of extracellular Cl− with gluconate− diminishes the inward tail current (Cl− efflux) at a membrane potential of −100 mV due to the lowered outward current (gluconate− influx) at membrane potential of 100 mV. These results suggest that the direction and rate of charge-carrier movement regulate the open time of mPanx1, and that the previously reported voltage-dependence of Panx1 channel gating is not directly mediated by the membrane potential but rather by the direction and amplitude of currents through the channel.

Transient receptor potential vanilloid 4 (TRPV4) channel as a target of crotamiton and its bimodal effects.

The sensation of itching can be defined as "an unpleasant cutaneous sensation that provokes a desire to scratch." The perception of itching is not critical for the maintenance of life, but persistent itching can be extremely irritating and decreases the quality of life. Crotamiton (N-ethyl-o-crotonotoluidide) has been used as an anti-itch agent for humans for around 70years. In spite of the long use of crotamiton, its mechanism of action remains unknown. We hypothesized that crotamiton might have effects on transient receptor potential (TRP) channels expressed in the peripheral nervous system and the skin. We first examined the effects of crotamiton on TRP channels by whole-cell patch-clamp recordings. We found that crotamiton strongly inhibited TRPV (vanilloid) 4 channels followed by large currents after crotamiton washout. In mice, crotamiton inhibited itch-related behaviors induced by a TRPV4-selective agonist (GSK1016790A). We biophysically investigated the large TRPV4 currents after crotamiton washout. Comparing single-channel open probabilities and current amplitudes of TRPV4, increases in both parameters were found to contribute to the large washout currents of TRPV4. Because the change in current amplitudes suggested pore dilation of TRPV4, we examined this possibility with cation replacement experiments and by measuring changes in reversal potentials. Greater cation influxes and changes in reversal potentials upon crotamiton washout were observed, suggesting that the TRPV4 pore dilated in its uninhibited state. From these results, we identified the molecular target of crotamiton as TRPV4 and demonstrated pore dilation of TRPV4 upon crotamiton washout.

Electrostatic tuning of the pre- and post-hydrolytic open states in CFTR.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that couples adenosine triphosphate (ATP) hydrolysis at its nucleotide-binding domains to gating transitions in its transmembrane domains. We previously reported that the charge-neutralized mutant R352C shows two distinct open states, O1 and O2 The two states could be distinguished by their single-channel current amplitudes: O1 having a smaller amplitude (representing a prehydrolytic open state) and O2 having a larger amplitude (representing a post-hydrolytic open state). In this study, a similar phenotype is described for two mutations of another pore-lining residue, N306D and N306E, suggesting that alterations of the net charge within CFTR's pore confer this unique conductance aberration. Because moving either of the two endogenous charges, R303 and R352, to positions further along TM5 and TM6, respectively, also results in this O1O2 phenotype, we conclude that the position of the charged residue in the internal vestibule affects hydrolysis-dependent conductance changes. Furthermore, our data show that the buffer and CFTR blocker morpholino propane sulfonic acid (MOPS-) occludes the O1 state more than it does the O2 state when the net charge of the internal vestibule is unchanged or increased. In contrast, when the net charge in the internal vestibule is decreased, the differential sensitivity to MOPS- block is diminished. We propose a three-state blocking mechanism to explain the charge-dependent sensitivity of prehydrolytic and post-hydrolytic open states to MOPS- block. We further posit that the internal vestibule expands during the O1 to O2 transition so that mutation-induced electrostatic perturbations within the pore are amplified by the smaller internal vestibule of the O1 state and thus result in the O1O2 phenotype and the charge-dependent sensitivity of the two open states to MOPS- block. Our study not only relates the O1O2 phenotype to the charge distribution in CFTR's internal vestibule but also provides a toolbox for mechanistic studies of CFTR gating by ATP hydrolysis.

Activation of the Human Epithelial Sodium Channel (ENaC) by Bile Acids Involves the Degenerin Site

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin ion channel family, which also includes the bile acid-sensitive ion channel (BASIC). So far little is known about the effects of bile acids on ENaC function. ENaC is probably a heterotrimer consisting of three well characterized subunits (αβγ). In humans, but not in mice and rats, an additional δ-subunit exists. The aim of this study was to investigate the effects of chenodeoxycholic, cholic, and deoxycholic acid in unconjugated (CDCA, CA, and DCA) and tauro-conjugated (t-CDCA, t-CA, t-DCA) form on human ENaC in its αβγ- and δβγ-configuration. We demonstrated that tauro-conjugated bile acids significantly stimulate ENaC in the αβγ- and in the δβγ-configuration. In contrast, non-conjugated bile acids have a robust stimulatory effect only on δβγENaC. Bile acids stimulate ENaC-mediated currents by increasing the open probability of active channels without recruiting additional near-silent channels known to be activated by proteases. Stimulation of ENaC activity by bile acids is accompanied by a significant reduction of the single-channel current amplitude, indicating an interaction of bile acids with a region close to the channel pore. Analysis of the known ASIC1 (acid-sensing ion channel) crystal structure suggested that bile acids may bind to the pore region at the degenerin site of ENaC. Substitution of a single amino acid residue within the degenerin region of βENaC (N521C or N521A) significantly reduced the stimulatory effect of bile acids on ENaC, suggesting that this site is critical for the functional interaction of bile acids with the channel.

Digitalized K(+) Occupancy in the Nanocavity Holds and Releases Queues of K(+) in a Channel.

The mechanisms of ion permeation through potassium channels have been extensively examined. Molecular dynamics (MD) simulations have demonstrated that rapidly permeating ions collide near the selectivity filter (SF) ("knock-on" mechanism), but this oversimplified mechanism is insufficient to account for the experimentally observed single-channel current amplitudes. Here, we analyzed the MD-simulated ion trajectories through a Kv1.2 potassium channel using an event-oriented analysis method, and surprisingly, we found that the nanocavity (NC) governs ion permeation in a digital fashion. The NC has a maximal diameter of 10 Å and stands between the intracellular bulk solution and the SF, which holds only up to one K(+) during permeation. Accordingly, the K(+) concentration in the intracellular solution is translated as a digitalized zero or one K(+) in the NC. When the ion number in the NC is zero, the multiple ions in the SF are mostly immobilized. By contrast, when the number of ions in the NC is one, the structured water in the NC mediates the ion-occupied status to the queueing ions in the SF, and the ions then initiate a collective outward motion. Accordingly, the one ion in the NC serves as a catalytic intermediate for permeation, which quantitatively accounts for the experimentally obtained conductance-concentration relationships. We conclude that the ion movements are coherent across the entire pore.

Regulation of Spermatogenic Cell T-Type Ca(2+) Currents by Zn(2+): Implications in Male Reproductive Physiology.

Zn(2+) is a trace metal which is important for spermatogenesis progression; its deficiency causes atrophy or malignant growth of the testis. Although testis, epididymis, and prostate contain high Zn(2+) concentrations, the molecular entities which are modulated by this metal are still under study. Interestingly, spermatogenic cells mainly express CaV 3.2-encoded T-type Ca(2+) currents (ICaT) which are positively or negatively modulated by Zn(2+) in other tissues. To explore whether ICaT could be regulated by Zn(2+) and albumin, its main physiological carrier, we performed whole cell electrophysiological recordings of spermatogenic cell ICaT in the absence or presence of different Zn(2+) concentrations. Zn(2+) decreased ICaT in a concentration-dependent manner (IC50 = 2 μM) and this inhibition could only be completely removed in presence of albumin. Differently to previous reports, ICaT did not show a tonic inhibition by Zn(2+) . Further analysis showed that Zn(2+) did not affect the voltage dependency or the kinetics of current activation, but right shifted the steady-state inactivation curve and slowed inactivation and deactivation kinetics. Recovery from inactivation was also altered. However, these apparent alterations in gating properties are not enough to explain the strong ICaT reduction. Using non-stationary fluctuation analysis, we found that Zn(2+) mainly reduced the number of available Ca(2+) channels without changing the single channel current amplitude. ICaT modulation by Zn(2+) could be relevant for spontaneous Ca(2+) oscillations during spermatogenesis and in pathophysiological conditions such as diabetes.

The Fifth Transmembrane Segment of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Its Anion Permeation Pathway.

Previous studies have identified several transmembrane segments (TMs), including TM1, TM3, TM6, TM9, TM11, and TM12, as pore-lining segments in cystic fibrosis transmembrane conductance regulator (CFTR), but the role of TM5 in pore construction remains controversial. In this study, we employed substituted cysteine accessibility methodology (SCAM) to screen the entire TM5 defined by the original topology model and its cytoplasmic extension in a Cysless background. We found six positions (A299, R303, N306, S307, F310, and F311) where engineered cysteines react to intracellular 2-sulfonatoethyl methanethiosulfonate (MTSES⁻). Quantification of the modification rate of engineered cysteines in the presence or absence of ATP suggests that these six residues are accessible in both the open and closed states. Whole-cell experiments with external MTSES⁻ identified only two positive positions (L323 and A326), resulting in a segment containing 11 consecutive amino acids, where substituted cysteines respond to neither internal nor external MTSES⁻, a unique feature not seen previously in CFTR's pore-lining segments. The observation that these positions are inaccessible to channel-permeant thiol-specific reagent [Au(CN)₂]⁻ suggests that this segment of TM5 between F311 and L323 is concealed from the pore by other TMs and/or lipid bilayers. In addition, our data support the idea that the positively charged arginine at position 303 poses a pure electrostatic action in determining the single-channel current amplitude of CFTR and the effect of an open-channel blocker glibencalmide. Collectively, we conclude that the cytoplasmic portion of CFTR's TM5 lines the pore. Our functional data are remarkably consistent with predicted structural arrangements of TM5 in some homology models of CFTR.

Engineered Ionizable Side Chains.

One of the great challenges of mechanistic ion-channel biology is to obtain structural information from well-defined functional states. In the case of neurotransmitter-gated ion channels, the open-channel conformation is particularly elusive owing to its transient nature and brief mean lifetime. In this Chapter, we show how the analysis of single-channel currents recorded from mutants engineered to contain single ionizable side chains in the transmembrane region can provide specific information about the open-channel conformation without any interference from the closed or desensitized conformations. The method takes advantage of the fact that the alternate binding and unbinding of protons to and from an ionizable side chain causes the charge of the protein to fluctuate by 1 unit. We show that, in mutant muscle acetylcholine nicotinic receptors (AChRs), this fluctuating charge affects the rate of ion conduction in such a way that individual proton-transfer events can be identified in a most straightforward manner. From the extent to which the single-channel current amplitude is reduced every time a proton binds, we can learn about the proximity of the engineered side chain to the lumen of the pore. And from the kinetics of proton binding and unbinding, we can calculate the side-chain's affinity for protons (pK a), and hence, we can learn about the electrostatic properties of the microenvironment around the introduced ionizable group. The application of this method to systematically mutated AChRs allowed us to identify unambiguously the stripes of the M1, M2 and M3 transmembrane α-helices that face the pore's lumen in the open-channel conformation in the context of a native membrane.

Probing the Structure and Function of TMC1 in Sensory Hair Cells using Mutagenesis and Cysteine Modification

Transmembrane channel-like genes 1 and 2 (Tmc1 and Tmc2) have recently been implicated as components of the sensory transduction channel in mammalian hair cells. Mice deficient in both Tmc1 and Tmc2 have complete loss of hearing and balance function and lack sensory transduction, despite intact hair bundles and tip-links (Kawashima et al., 2011). Mice that express only TMC1 or TMC2 have distinct single-channel conductances and calcium selectivity. A methionine-to-lysine substitution at position 412 in TMC1 reduces the single-channel current amplitude and calcium permeability of transduction (Pan et al., 2013). These results suggest that TMCs participate as essential components of the sensory transduction channel in auditory and vestibular hair cells, but their exact role is not yet clear. They may form a vestibule at the mouth of the pore, the pore of the ion channel itself or both (Holt et al., 2014). Alternatively, TMCs may function as non-essential accessory subunits (Beurg et al., 2014). Progress toward understanding the structure and function of TMC proteins has been limited by the lack of homology to known genes or domains (Kurima et al., 2003); the low quantity of native protein (50-100 functional channels/cell); and poor membrane localization in heterologous cells. To circumvent these limitations, we combined electrophysiological recording with mutagenesis and cysteine modification in native hair cells. To probe TMC1 structure and function, mutant Tmc1 sequences were introduced, via viral transfection, into organotypic cultures harvested from Tmc1/Tmc2 doubly-deficient mice. We find that the mutant constructs restore sensitivity to hair bundle deflections in virally-transfected hair cells and that acute application of cysteine modification reagents alters the biophysical properties of sensory transduction within seconds. The data further support a direct role for TMC1 in sensory transduction in mammalian hair cells.

State-dependent blocker interactions with the CFTR chloride channel: implications for gating the pore

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is subject to voltage-dependent open-channel block by a diverse range of cytoplasmic anions. However, in most cases the ability of these blocking substances to influence the pore opening and closing process has not been reported. In the present work, patch clamp recording was used to investigate the state-dependent block of CFTR by cytoplasmic Pt(NO2)4(2-) ions. Two major effects of Pt(NO2)4(2-) were identified. First, this anion caused fast, voltage-dependent block of open channels, leading to an apparent decrease in single-channel current amplitude. Secondly, Pt(NO2)4(2-) also decreased channel open probability due to an increase in interburst closed times. Interestingly, mutations in the pore that weakened (K95Q) or strengthened (I344K, V345K) interactions with Pt(NO2)4(2-) altered blocker effects both on Cl(-) permeation and on channel gating, suggesting that both these effects are a consequence of Pt(NO2)4(2-) interaction with a single site within the pore. Experiments at reduced extracellular Cl(-) concentration hinted that Pt(NO2)4(2-) may have a third effect, possibly increasing channel activity by interfering with channel closure. These results suggest that Pt(NO2)4(2-) can enter from the cytoplasm into the pore inner vestibule of both open and closed CFTR channels, and that Pt(NO2)4(2-) bound in the inner vestibule blocks Cl(-) permeation as well as interfering with channel opening and, perhaps, channel closure. Implications for the location of the channel gate in the pore, and the operation of this gate, are discussed.

GABAA Receptor α and γ Subunits Shape Synaptic Currents via Different Mechanisms

Synaptic GABAA receptors (GABAARs) mediate most of the inhibitory neurotransmission in the brain. The majority of these receptors are comprised of α1, β2, and γ2 subunits. The amygdala, a structure involved in processing emotional stimuli, expresses α2 and γ1 subunits at high levels. The effect of these subunits on GABAAR-mediated synaptic transmission is not known. Understanding the influence of these subunits on GABAAR-mediated synaptic currents may help in identifying the roles and locations of amygdala synapses that contain these subunits. Here, we describe the biophysical and synaptic properties of pure populations of α1β2γ2, α2β2γ2, α1β2γ1 and α2β2γ1 GABAARs. Their synaptic properties were examined in engineered synapses, whereas their kinetic properties were studied using rapid agonist application, and single channel recordings. All macropatch currents activated rapidly (<1 ms) and deactivated as a function of the α-subunit, with α2-containing GABAARs consistently deactivating ∼10-fold more slowly. Single channel analysis revealed that the slower current decay of α2-containing GABAARs was due to longer burst durations at low GABA concentrations, corresponding to a ∼4-fold higher affinity for GABA. Synaptic currents revealed a different pattern of activation and deactivation to that of macropatch data. The inclusion of α2 and γ1 subunits slowed both the activation and deactivation rates, suggesting that receptors containing these subunits cluster more diffusely at synapses. Switching the intracellular domains of the γ2 and γ1 subunits substantiated this inference. Because this region determines post-synaptic localization, we hypothesize that GABAARs containing γ1 and γ2 use different mechanisms for synaptic clustering.

Paradoxical One-ion Pore Behavior of the Long β-Helical Peptide of Marine Cytotoxic Polytheonamide B

The cytotoxic 48-mer peptide, polytheonamide B (pTB), from a marine sponge forms a β6.3-helix with an inner diameter of 4 Å and a length of 45 Å, features that allow the selective permeation of monovalent cations across targeted cell membranes. To characterize this long, narrow pore, electrophysiological examination using a planar lipid bilayer method was performed. The single-channel current amplitude exhibited saturation for concentrated Cs+ or K+ solution, and the reversal potential in mixed solutions did not exhibit any anomalous mole-fraction behavior. These results suggest the one-ion permeation mechanism. This is in contrast to the short (26 Å) β6.3-helical gramicidin channel, which holds two ions simultaneously. The paradoxical one-ion permeation through the long pTB channel was modeled with a discrete-state Markov model. Ions permeate through the channel by stepping between two binding sites in the pore, but never occupy these sites simultaneously in either pure or mixed ion solution.

Functional Properties of the Retinal Glutamate Transporters GLT-1c and EAAT5

In the mammalian retina, glutamate uptake is mediated by members of a family of glutamate transporters known as "excitatory amino acid transporters (EAATs)." Here we cloned and functionally characterized two retinal EAATs from mouse, the GLT-1/EAAT2 splice variant GLT-1c, and EAAT5. EAATs are glutamate transporters and anion-selective ion channels, and we used heterologous expression in mammalian cells, patch-clamp recordings and noise analysis to study and compare glutamate transport and anion channel properties of both EAAT isoforms. We found GLT-1c to be an effective glutamate transporter with high affinity for Na(+) and glutamate that resembles original GLT-1/EAAT2 in all tested functional aspects. EAAT5 exhibits glutamate transport rates too low to be accurately measured in our experimental system, with significantly lower affinities for Na(+) and glutamate than GLT-1c. Non-stationary noise analysis demonstrated that GLT-1c and EAAT5 also differ in single-channel current amplitudes of associated anion channels. Unitary current amplitudes of EAAT5 anion channels turned out to be approximately twice as high as single-channel amplitudes of GLT-1c. Moreover, at negative potentials open probabilities of EAAT5 anion channels were much larger than for GLT-1c. Our data illustrate unique functional properties of EAAT5, being a low-affinity and low-capacity glutamate transport system, with an anion channel optimized for anion conduction in the negative voltage range.