Channel Conductance Research Articles

A vibrational spectroscopic approach to elucidate the molecular mechanism behind the channel activity of viroporins.

The prevalence of diseases caused by pathogenic viruses and the scarcity of treatment and vaccination options call for extensive research of the molecular mechanisms underlying the function of these viruses. Small hydrophobic viral proteins with ion channel activity, so-called viroporins, are involved in the virus particle entry into and release from the host cell. One of these viroporins, the Influenza A M2 channel, has shown to be the target for anti-viral drugs motivating spectroscopic investigations of other viroporins to understand their relevance as potential anti-viral treatment targets. We used the Influenza A M2 viroporin as a model system to establish a vibrational spectroscopic approach to examine the gating mechanism and proton conductance of this pH-activated proton channel. In this work, we immobilized reconstituted M2 on a self-assembled monolayer bound to a nanostructured gold film that can be used for surface-enhanced infrared absorption (SEIRA) spectroscopy. Employing pH-induced SEIRA difference spectroscopy we monitor protonation events and changes in the α-helix amide I absorption due to a pH-dependent channel opening that leads to proton conductivity. Furthermore, the surface selection rule of SEIRA enabled us to observe the reorientation of the α-helical structures associated with the channel opening in-situ. Combining the spectroscopic data with computational vibrational spectroscopic calculations we model the large-scale reorganization of M2's α-helices, and by this quantify the opening angle of the channel transitioning from closed to the activated state. We aim to utilize this approach to enable a combined structural and functional analysis of viroporins of current relevance.

The conductance of TMC1-containing mechanotransducer channels and the roles of LHFPL5 and TMIE in cochlear hair cells.

Transmembrane channel-like protein 1 (TMC1) is thought to form the ion-conducting pore of the mechanotransducer (MET) channel in auditory hair cells, assisted by accessory subunits TMIE, LHFPL5 and CIB2. LHFPL5 is most likely connected to the carboxy terminus of PCDH15 at the lower end of the tip link. The organization of the channel complex is still unclear, but a TMC1 structure based on homology with TMEM16A indicates ten transmembrane (TM) domains with the pore lined by TMs 4-7. Using single channel analysis and ionic permeability measurements, we characterized six missense mutations in the purported pore region of mouse TMC1. MET currents could be recorded in cochlear hair cells of neonatal mutant mice, showing all mutations reduced the Ca2+ permeability of the channel. In addition, Tmc1 p.E520Q and Tmc1 p.D528N reduced channel conductance whereas Tmc1 p.W554L and Tmc1 p.D569N lowered channel expression without affecting the conductance. The consequences of these mutations endorse TMC1 as the pore of the MET channel, the mutations occurring in transmembrane domains 6-7. The accessory subunits LHFPL5 and TMIE are thought to be involved in targeting TMC1 to the tips of the stereocilia. We found sufficient expression of TMC1 in hair cells of Lhfpl5 and Tmie knockout mice to characterize the remaining MET channels, which could still be gated by hair bundle deflections. Single-channel conductance was unaffected in Lhfpl5-/- but was reduced in Tmie‑/-, implying TMIE very likely contributes to the pore. Both the working range and half-saturation point of the residual MET current in Lhfpl5-/- were substantially increased, suggesting that LHFPL5 is part of the mechanical coupling between the tip-link and the MET channel.

Fine-structure constant and hysteresis regimes in the giant squid propagating action potential.

We present a phase space based phenomenological theory for the steady propagation of the action potential using the Rosenthal-Bezanilla experimental data. Time derivatives of giant squid action potential, its velocity of propagation, the radius of the axon and its capacitance, and the resistivity of axoplasm, yield the total ionic, capacitive, and membrane currents of the charge conserving cable equation. Evidence is presented that the sodium channels lattice has a polarization flip at the inception of the action potential and the corresponding flip reversal at the peak of the action potential while traversing a ferroelectric hysteresis loop. The polarization flip at the inception of the action potential is followed by capacitive polarization current known as gating current. The polarization flip at the peak of the action potential is preceded by a small sodium polarization current associated with sodium current inactivation. Ionic currents and sodium polarization ionic currentsare taken to have the familiar structure, as the product of maximum conductance, driving force, and fraction of open channels. Fractions of open channels are fitted in the lab frame by modified Avrami equations seeded with the value of the fine-structure constant α = 0.0072973... . The existence of sodium channels domains with two different symmetries, albeit only one being stable in squid axon, suggests the possibility of neurons with at least 2 stable states, the necessary condition for storage and retrieval of memories. It is expected that presented results will provide the framework for further analysis of thermodynamic phase changing behavior, the role of quantum mechanics in the flow of ions trough ionic channels and in particular the role of ferroelectric sodium channels lattice behavior in storage and retrieval of memories and nerve excitability.

Water movement in voltage-gated proton channel (HV1).

To date, the permeation mechanism of the Hv1 channel has been controversial, due to the lack of an open structure. Besides the selectivity filter (SF), several studies have shown that the highly conserved S4 asparagine is important for the channel's proton conduction. The protons’ conduction mechanism cannot be separated from the nature of water; thus, using electrophysiological techniques, we evaluated how different mutations directed to the N264 site of the CiHv1 channel affect the proton conduction properties, and these results were contrasted with classical MD simulations in which we explore how the hydration profiles, and the dipole angle of H2O molecules along the protein change with the different N264 mutations. We observed that the super-conductive N264E construct has a favorable electrostatic profile and the H2O molecules near the SF had more freedom of movement and showed more dipole angle configurations, whereas low-conductive N264R had an unfavorable electrostatic profile and a virtually dry zone near the SF. Hv1-injected X. laevis oocytes showed an increased osmotic permeability coefficient. These experiments suggest the presence of water fluxes though the Hv1 channel. With the results of this study, we were able to increase the understanding on the H+ conduction in Hv1 channels and we proposed a new model that takes into consideration water movement. Thus, in our model we propose that the Hv1 conduction possesses two main components: (1) the H2O configurations along the protein and (2) the electrostatic profile, and the proton permeation mechanism occurs in a non-Grotthuss manner, probably accompanied by water fluxes at least at higher voltage structure configurations.

Site-specific phosphorylation of VDAC1 decreases its susceptibility to oligomerization.

The voltage-dependent anion channel 1 (VDAC1) is the primary pathway for metabolite and ion flux across the outer mitochondrial membrane. Oligomerization of VDAC1 that results in the formation of a mega-pore is a critical step in the release of cytochrome c and mitochondrial DNA that triggers apoptosis and inflammasome activation, respectively. However, the underlying mechanism that leads to mega-pore formation is unresolved. Previously, we identified phosphorylation of VDAC1 at S35 to be associated with cardioprotection. Here we report a novel finding that S35 phosphorylation regulates VDAC1 oligomerization and, subsequently, mega-pore formation. In 90 min recordings of channel activity of recombinant VDAC1 in planar lipid bilayers, wild-type (WT) VDAC1 exhibited cumulative current increases due to multiple channel insertions. In 50% of the recordings, a dramatic increase in cumulative channel conductance was observed (>1.8 µS). This was accompanied by a progressive shift at baseline, suggestive of high local ionic accumulation at the bilayer surface. These distinct biophysical characteristics were attributed to mega-pore formation. The probability of similar cumulative large conductances with associated baseline shifts was reduced in the phosphomimetic S35E VDAC1 (20% vs 50% in WT), while it was increased in a non-phosphorylatable mutant S35A (90% vs 50% in WT). In VDAC1 knock-out H9c2 cells expressing WT or mutant (S35A/S35E) VDAC1, 400 µM H2O2 induced the formation of VDAC1 oligomers. However, compared to WT, the formation of trimers and higher order oligomers was significantly decreased in cells expressing the S35E mutant, while it was significantly increased in cells expressing the S35A mutant. These effects are consistent with the corresponding biophysical characteristics observed in the bilayer experiments. Our results suggest that phosphorylation of S35 decreases, while dephosphorylation increases, the susceptibility of VDAC1 to oligomerization and mega-pore formation.

The energetics of silent translocation through a biological nanopore.

Electrophoretically driving molecules through the lumen of a nanopore can produce transient channel conductance fluctuations by occlusion of ionic flow. However, many molecules do not possess the necessary size, shape, charge, or binding affinity to produce a detectable electrical signature. For cases where transport is quick (e.g., <20 μs), or transport is exceedingly rare, or critical interaction with the pore is lacking, the resulting current recordings appear silent. Within the performance range of common electrophysiological amplifiers, these recordings show no excess noise and no momentary electrical blockage. In general, there is no electrical way to discern if translocation occurs. Here, we report on the electrically silent passage of fluorescent dye molecules through the alpha hemolysin (αHL) nanopore. Simultaneous single-molecule fluorescence and electrical measurements in bilayers formed on microcavity electrode arrays (MECA-opto chips) demonstrate that translocation does, indeed, take place, albeit infrequently. We describe the methodology for detecting fluorophore transport, report observations of silent translocations as a function of time, dye concentration, and nanopore population in the bilayer. Lastly, measurements of the translocation rate as a function of applied potential permit estimation of the energy barrier for transport through the pore, as well as the effective charge on the dye. The energetics of silent translocation can only be revealed using measurement techniques that complement traditional electrical recording.

Characterization of human AQP5 mutations found in patients suffering from palmoplantar keratosis (Bothnian type).

Aquaporins are structurally well conserved within all kingdoms of life and can be divided into two main groups: (i) orthodox aquaporins conducting only water molecules, and (ii) aquaglyceroporins channeling water and other small uncharged solutes. Water transport through these integral membrane channels takes place in a single-file fashion through the pores of each of the protomers that form tetrameric units in cell membranes.[PP1] Here we focus on the functional consequences of mutations in the orthodox aquaporin-5 (Aqp5), commonly expressed in salivary glands, digestive and respiratory systems, kidneys, the CNS, and sweat glands. These mutations cause hereditary palmoplantar keratoma (Bothnia type; PPKB), marked by hyperkeratotic lesions on the surface of palms and soles. They differ in the positions of the substituted amino acids as well as backbone interactions. We overexpressed some of the mutations in S. cerevisiae as YFP-fusion proteins, purified and reconstituted the water channels into lipid vesicles, and monitored osmotic vesicle deflation under stopped-flow conditions. Additionally assessing the number of reconstituted channels by fluorescence correlation spectroscopy allowed calculation of unitary water channel conductances. The significance of the alterations in water channeling capabilities is discussed with respect to disease genesis.

A novel activator of Piezo1: Electrophysiological study.

Mechanosensitive ion channel Piezo1 plays important roles in diverse physiological processes, including differentiation, angiogenesis, vasodilation, wound healing, and inflammation. To date, three Piezo1 activators (Yoda1, Jedi1, and Jedi2) were discovered through high-throughput screening. These are artificially synthesized chemicals and not pharmacologically authorized, thus having potentials to cause problems when being administrated in human. In this study, we introduce a novel activator of Piezo1 (NAP1) that is a natural component of organisms. The effect of NAP1 on Piezo1 current was tested through electrophysiological recording on Piezo1-deficient (P1KO) HEK293T cells with or without transfection of human Piezo1 (hPiezo1). Without hPiezo1 on P1KO HEK293T, mechanical stimulation failed to activate current, which was still not observable after the application of NAP1 to the cells. However, NAP1 sensitized hPieoz1 currents in various patch configurations. In cell-attached recording, extracellular NAP1 (10, 30, and 100 µM) increased hPiezo1 currents in a dose-dependent manner, shifted the current-stimulus curve leftwards, and increased the pressure sensitivity. However, 100 µM NAP1 didn’t alter the single channel conductance of hPiezo1. In outside-out and whole-cell recordings, NAP1 reversibly enhanced hPiezo1 currents. The association and dissociation times of 100 µM NAP1 measured in the outside-out configuration were approximately 8 seconds. Like Yoda1, NAP1 did not sensitize human Piezo2 in the whole cell configuration. Since NAP1 is biocompatible and orally administrable, it will replace the other Piezo1 activators, with its biocompatibility triggering the vitalization of animal studies involving Piezo1.

Lipid dependence of BsYetJ calcium transport activity by fluorescence and ESR techniques.

Recent studies based on X-ray crystallography and pulsed electron spin resonance results have revealed substantial structural insights for the mechanistic understanding of the Ca2+ transport mechanism in the membrane protein YetJ from Bacillus subtilis (BsYetJ). One striking finding is that its structure-function relationship is sensitive to the difference between the surrounding environments, detergent versus POPC lipid. Here, we continue on the study to explore the molecular details of BsYetJ in response to changes in lipid composition, at various mixtures of PC, PE, PI, and cholesterol. Using mutagenesis and fluorescence-based functional assays, we investigate several important negatively charged residues in the BsYetJ and reveal how the interplay determines the calcium channel conductance. This study identifies the conformations of BsYetJ in a complex lipid environment using electron spin resonance and molecular dynamics. Together with the functional data, we propose a mechanism, composed of previously unidentified selectivity filters, for the calcium transport activity of BsYetJ. We show that the calcium transport activity of BsYetJ can be modulated by lipid compositions. Our study provides new insights into the structural impact of the mutations and the development of the mechanistic model of BsYetJ.

Emergent pacemaking and tissue heterogeneity in a calcium feedback regulatory model of the sinoatrial node.

The sinoatrial node (SAN) is the primary pacemaker of the heart. SAN activity emerges at an early point in life, and maintains a steady rhythm for the lifetime of the organism. The ion channel composition and currents of the cells can be influenced by a variety of factors. Therefore, the emergent activity and long-term stability imply some form of dynamical feedback control of the SAN cell activity. Here, we adapt a recent neuronal model to the SAN rabbit cell. The model describes a minimal regulatory mechanism of neuronal ion channel conductance based on a feedback loop defined by an intracellular [Ca2+] level as its target. Briefly, the cell upregulates or downregulates its channel mRNA and membrane expression levels based on the difference between the intercellular Ca2+ level in the cell and a set intercellular Ca2+ target. Based on this feedback model, spontaneous electrical activity emerges in the SAN cell from a quiescent state with low initial conductances. As conductances increase, the intracellular [Ca2+] level reaches the target, and ion channel conductance reach a steady state consistent with sustained spontaneous activity. In a 2D tissue, variability in [Ca2+] target leads to heterogeneous ion channel expression and Ca2+ transients throughout the tissue. Further, dominant focal clusters appear, which interact with one another leading to a heterogeneous tissue cycle length, implying that variability in heart rate is an emergent property of the feedback model. Finally, the 2D tissue is robust to the silencing of leading cells or ion channel knock-outs. Thus, the calcium feedback regulatory model explains a number of experimental data using a minimal description of intracellular calcium and ion channel regulatory networks.

Nanofluidics: Transport in chirality-controlled carbon nanotube porins (CNTPs) and fluorescence ultrashort nanotubes (FUNs).

Carbon nanotubes (CNTs) with different chirality are great models to study the influence of channel electronic properties on ion transport through nanofluidic channels. We prepared short chirality-separated pure (6,5), (7,4), and (7,5)/(8,4) CNT porins (CNTPs), which have different electronic properties but are almost identical in diameter. Single channel conductance from potassium transport through these CNTPs indicates that electronic properties have a weak effect on ion transport. Sodium and lithium ion transport through different chirality CNTPs have also been studied. Chemical functionalities and defects on the CNTP walls can also influence transport properties. To address that possibility, we measured ion transport through fluorescence ultrashort nanotubes (FUNs) - functionalized channels with defective walls dispersed by surfactants such as CHAPS and SDOC. These channels have significantly lower conductance compared to the pristine (6,5) CNTPs. Finally, we discuss other factors that influence conductance in these nanofluidic channels.

Physiological role of SK channels in modulating cardiac repolarization: APD and dispersion at slow heart rate in long QT syndrome.

Long QT syndrome (LQTS) is a congenital disease associated with polymorphic ventricular tachycardia (pVT) and sudden cardiac death. The hallmarks of LQTS are early afterdepolarizations that initiate pVT and dispersion of repolarization allowing reentry formation. Small conductance Ca2+-activated K+ (SK) channels are directly gated by Ca2+ and provide feedback from Ca2+ to membrane voltage to accelerate cardiac repolarization, which may help normalizing LQTS. Using optical mapping, we examined the role of ISK in transgenic rabbit models of LQT1, LQT2, and LQT5 and found that enhancing ISK with NS309 consistently shortened APD, reduced APD dispersion, and suppressed pVT genesis universally in the three LQT rabbit models. Enhancement of ISK has a greater impact at longer cycle length, flattening restitution curves at slow heart rate where most pVTs occur in LQTS. We incorporated a six state ISK gating simulation combined with intrinsic rectification and divalent block into the Mahajan rabbit action potential model to investigate the role of ISK in suppressing LQT-related arrhythmias. Increasing levels of SK channel conductance (gSK) were able to shorten and reduce APD dispersion simulated by conductance gradients of gKs by ∼64% under LQT2 conditions and gtof by ∼70% in LQT1 conditions. Computer modeling indicates that ISK can reduce APD dispersion through two mechanisms; increased Ca2+ duration in the prolonged APD cells augment ISK to shorten long APD and lowering plateau Vm by ISK masks IKs heterogeneity by reducing IKs's role in repolarization. In conclusion, the Ca2+-dependent gating of ISK allows it to directly feedback on the APD especially when other K+ channels are not functional and to be a potential therapeutic target for LQTS.

The bisphosphonate zoledronic acid is a TRPV1 channel inhibitor.

The heat and capsaicin sensor, the transient receptor potential vanilloid type 1 (TRPV1) channel, is critically involved in pain sensation. Using compounds that activate TRPV1, such as capsaicin (CAP), produces analgesic effects. Hence the channel is a promising target for the management of pain conditions. However, when TRPV1 channels are sensitized in neuropathic pain conditions, inhibitors could be a better option for pain management. We have obtained evidence supporting that the bisphosphonate compound zoledronic acid (ZOL) acts as an inhibitor of the TRPV1 channel. ZOL is typically used in the treatment of bone diseases reducing bone mass loss, deterioration in bone microarchitecture and fracture risk, having as a collateral effect a decrease in pain sensation. We have observed, expressing TRPV1 in Xenopus laevis oocytes followed by patch-clamp recordings in inside-out configuration, that ZOL at doses ranging from 1 nM up to 30 μM reduces channel conductance and inhibits the activity of the channel deeply. Only high doses of CAP (≥ 10 μM) allow for recover of TRPV1 currents after ZOL treatment. Molecular modeling followed by docking analysis suggests that ZOL could potentially bind to TRPV1 at the vanilloid pocket and the PI(4,5)P2 binding site. Furthermore, like capsazepine, ZOL (100 nM) is able to eliminate the depression observed by applying CAP (1 μM) on synaptic transmission in the dentate gyrus. In addition, when ZOL is use during the induction of synaptic plasticity mediated by TRPV1, it eliminates plasticity. The compound did not affect the endocannabinoid production machinery suggesting that the ZOL is acting specifically on TRPV1 to hinder plasticity. Together, our evidence strongly supports that ZOL can inhibit TRPV1 activity.

A novel approach to dissect ions occupancy and dissociation constant of individualbinding sites within the K+-channel selectivity filter.

K+-channels permeate Rb+ as well as they do K+, although with a lower single channel conductance. KcsA crystal structure displays four K+ bound to its selectivity filter (SF); however, when crystallized in Rb+, ions were only found at the binding sites 1, 3, and 4. This observation explains the lower single conductance of K+-channels in rubidium. We reported a KcsA mutant (G77A) that stabilized the SF in the 2-4 ion bound configuration with K+ ions coordinated in the 2nd and 4th binding sites. Crystallizing the KcsA-G77A structure in rubidium interestingly showed Rb+ ions bound to the 1st and 4th binding sites. Like in the wild-type channel, the 2nd binding site was uncapable of binding rubidium. Therefore, we used the G77A to measure the 1st binding site dissociation constant for Rb+ by Isothermal Titration Calorimetry (ITC). Previously, computational studies have suggested that: 1) the SF's 4th ion binding site can be occupied by K+ or Na+ and hence it should not contribute to the SF's K+ binding affinity and 2) the 2nd binding site is responsible for the K+ binding affinity displayed by K+-channels SF. In here, we are showing that in K+-channels the 2nd binding site is indeed responsible for K+ binding affinity while the 1st site is responsible for the Rb+ ion binding affinity, since the G77A mutant (a simplified structural model for the SF) showed a wild-type like Rb+ dissociation constant. Therefore, we propose that in K+-channels the 1st ion binding site is responsible for the high rubidium over sodium selectivity shown by KcsA and potentially other potassium channels. Our advances in applying this approach for the alkali metal ion series will be presented.

Investigation of factors that influence the temperature sensitivity of the TRPM2 cation channel.

TRPM2 is a cation channel gated by Ca2+, ADPR and PiP2. The channel plays important role in the central and peripheral heat detection. To understand the physiological relevance of the channel in the regulation of body temperature we conducted electrophysiological studies in cell free patches at various temperatures and agonist concentrations. In the temperature range of 15-40°C we found that heating alone cannot open the channel, gating is strictly agonist dependent. Rising temperature increases channel conductance, open probability and apparent agonist binding affinities. We identified a strongly temperature dependent positive feedback on channel activity caused by the Ca2+ influx through the channel's pore. This temperature dependent Ca2+ influx generates a supraphysiological intracellular [Ca2+] nanodomain that tunes channel activity. These findings explain the physiological relevance of the channel in the regulation of body temperature. Based on our measured data we constructed a mechanistic gating model that allows the calculation of agonist concentrations in the channel's direct environment from measured open probabilities at given temperatures. This model allows us to further investigate the TRPM2 channel's temperature dependent function in different cellular environments and study how the resting membrane potential or the intracellular Ca2+ buffering properties modulate the channel's temperature threshold in different cell types.

Molecular dynamics study of Cl− permeation through cystic fibrosis transmembrane conductance regulator (CFTR)

The recent elucidation of atomistic structures of Cl− channel CFTR provides opportunities for understanding the molecular basis of cystic fibrosis. Despite having been activated through phosphorylation and provided with ATP ligands, several near-atomistic cryo-EM structures of CFTR are in a closed state, as inferred from the lack of a continuous passage through a hydrophobic bottleneck region located in the extracellular portion of the pore. Here, we present repeated, microsecond-long molecular dynamics simulations of human CFTR solvated in a lipid bilayer and aqueous NaCl. At equilibrium, Cl− ions enter the channel through a lateral intracellular portal and bind to two distinct cationic sites inside the channel pore but do not traverse the narrow, de-wetted bottleneck. Simulations conducted in the presence of a strong hyperpolarizing electric field led to spontaneous Cl− translocation events through the bottleneck region of the channel, suggesting that the protein relaxed to a functionally open state. Conformational changes of small magnitude involving transmembrane helices 1 and 6 preceded ion permeation through diverging exit routes at the extracellular end of the pore. The pore bottleneck undergoes wetting prior to Cl− translocation, suggesting that it acts as a hydrophobic gate. Although permeating Cl− ions remain mostly hydrated, partial dehydration occurs at the binding sites and in the bottleneck. The observed Cl− pathway is largely consistent with the loci of mutations that alter channel conductance, anion binding, and ion selectivity, supporting the model of the open state of CFTR obtained in the present study.

Intermediate conductance Ca2+-activated potassium channels are activated by functional coupling with stretch-activated nonselective cation channels in cricket myocytes.

Cooperative gating of localized ion channels ranges from fine-tuning excitation-contraction coupling in muscle cells to controlling pace-making activity in the heart. Membrane deformation resulting from muscle contraction activates stretch-activated (SA) cation channels. The subsequent Ca2+ influx activates spatially localized Ca2+-sensitive K+ channels to fine-tune spontaneous muscle contraction. To characterize endogenously expressed intermediate conductance Ca2+-activated potassium (IK) channels and assess the functional relevance of the extracellular Ca2+ source leading to IK channel activity, we performed patch-clamp techniques on cricket oviduct myocytes and recorded single-channel data. In this study, we first investigated the identification of IK channels that could be distinguished from endogenously expressed large-conductance Ca2+-activated potassium (BK) channels by adding extracellular Ba2+. The single-channel conductance of the IK channel was 62 pS, and its activity increased with increasing intracellular Ca2+ concentration but was not voltage-dependent. These results indicated that IK channels are endogenously expressed in cricket oviduct myocytes. Second, the Ca2+ influx pathway that activates the IK channel was investigated. The absence of extracellular Ca2+ or the presence of Gd3+ abolished the activity of IK channels. Finally, we investigated the proximity between SA and IK channels. The removal of extracellular Ca2+, administration of Ca2+ to the microscopic region in a pipette, and application of membrane stretching stimulation increased SA channel activity, followed by IK channel activity. Membrane stretch-induced SA and IK channel activity were positively correlated. However, the emergence of IK channel activity and its increase in response to membrane mechanical stretch was not observed without Ca2+ in the pipette. These results strongly suggest that IK channels are endogenously expressed in cricket oviduct myocytes and that IK channel activity is regulated by neighboring SA channel activity. In conclusion, functional coupling between SA and IK channels may underlie the molecular basis of spontaneous rhythmic contractions.

Structural Mechanism of Ionic Conductivity of the TRPV1 Channel

The so-called “hydrophobic gating” is widely discussed as a putative mechanism to control water and ion conduction via ion channels. This effect can occur in narrow areas of the channels pore lined by non-polar residues. In the closed state of the channel, such regions may spontaneously transit to a dehydrated state to block water and ions transport without full pore occlusion. In the open state, the hydrophobic gate is wide enough to provide sustainable hydration and conduction. Apparently, the transport through the open hydrophobic gate may by facilitated by some polar residues that assist polar/charged substances to overcome the energy barrier created by nonpolar environment. In this work, we investigated the behavior of Na+ ions and their hydration shells in the open pore of the rat TRPV1 ion channel by molecular dynamics simulations. We show that polar protein groups coordinate water molecules in such a way as to restore the hydration shell of ions in the hydrophobic gate that ensures ion transport through the gate in a fully hydrated state.

Additive Potentiation of R334W-CFTR Function by Novel Small Molecules.

The R334W (c.1000C>T, p.Arg334Trp) is a rare cystic fibrosis (CF)-causing mutation for which no causal therapy is currently approved. This mutation leads to a significant reduction of CF transmembrane conductance regulator (CFTR) channel conductance that still allows for residual function. Potentiators are small molecules that interact with CFTR protein at the plasma membrane to enhance CFTR-dependent chloride secretion, representing thus pharmacotherapies targeting the root cause of the disease. Here, we generated a new CF bronchial epithelial (CFBE) cell line to screen a collection of compounds and identify novel potentiators for R334W-CFTR. The active compounds were then validated by electrophysiological assays and their additive effects in combination with VX-770, genistein, or VX-445 were exploited in this cell line and further confirmed in intestinal organoids. Four compounds (LSO-24, LSO-25, LSO-38, and LSO-77) were active in the functional primary screen and their ability to enhance R334W-CFTR-dependent chloride secretion was confirmed using electrophysiological measurements. In silico ADME analyses demonstrated that these compounds follow Lipinski’s rule of five and are thus suggested to be orally bioavailable. Dose−response relationships revealed nevertheless suboptimal efficacy and weak potency exerted by these compounds. VX-770 and genistein also displayed a small potentiation of R334W-CFTR function, while VX-445 demonstrated no potentiator activity for this mutation. In the R334W-expressing cell line, CFTR function was further enhanced by the combination of LSO-24, LSO-25, LSO-38, or LSO-77 with VX-770, but not with genistein. The efficacy of potentiator VX-770 combined with active LSO compounds was further confirmed in intestinal organoids (R334W/R334W genotype). Taken together, these molecules were demonstrated to potentiate R334W-CFTR function by a different mechanism than that of VX-770. They may provide a feasible starting point for the design of analogs with improved CFTR-potentiator activity.

Simulation-predicted and -explained inheritance model of pathogenicity confirmed by transgenic mice models

Variants in the gap junction beta-2 (GJB2) gene are the most common cause of hereditary hearing impairment. However, how GJB2 variants lead to local physicochemical and structural changes in the hexameric ion channels of connexin 26 (Cx26), resulting in hearing impairment, remains elusive. In this study, using molecular dynamics (MD) simulations, we showed that detached inner-wall N-terminal “plugs” aggregated to reduce the channel ion flow in a highly prevalent V37I variant in humans. To examine the predictive ability of the computational platform, an artificial mutant, V37M, of which the effect was previously unknown in hearing loss, was created. Microsecond simulations showed that homo-hexameric V37M Cx26 hemichannels had an abnormal affinity between the inner edge and N-termini to block the narrower side of the cone-shaped Cx26, while the most stable hetero-hexameric channels did not. From the perspective of the conformational energetics of WT and variant Cx26 hexamers, we propose that unaffected carriers could result from a conformational predominance of the WT and pore-shrinkage-incapable hetero-hexamers, while mice with homozygous variants can only harbor an unstable and dysfunctional N-termini-blocking V37M homo-hexamer. Consistent with these predictions, homozygous V37M transgenic mice exhibited apparent hearing loss, but not their heterozygous counterparts, indicating a recessive inheritance mode. Reduced channel conductivity was found in Gjb2V37M/V37M outer sulcus and Claudius cells but not in Gjb2WT/WT cells. We view that the current computational platform could serve as an assessment tool for the pathogenesis and inheritance of GJB2-related hearing impairments and other diseases caused by connexin dysfunction.