Electromechanics of lipid-modulated gating of potassium channels

Experimental studies reveal that the anionic lipid phosphatidic acid (POPA), non-phospholipid cholesterol, and cationic lipid DOTAP inhibit the gating of voltage-sensitive potassium (Kv) channels. Here, we develop a continuum electromechanical model to investigate the interaction of these lipids with the ion channel. Our model suggests that: (i) POPA lipids may restrict the vertical motion of the voltage-sensor domain through direct electrostatic interactions; (ii) cholesterol may oppose the radial motion of the pore domain of the channel by increasing the mechanical rigidity of the membrane; and (iii) DOTAP can reduce the effect of electrostatic forces by regulating the dielectric constant at the channel–lipid interface. The electromechanical model predictions for the three lipid types match well with the experimental observations and provide mechanistic insights into lipid-dependent gating of Kv channels.

Exploring Kv1.2 Channel Inactivation Through MD Simulations and Network Analysis

The KCNA2 gene encodes the Kv1.2 channel, a mammalian Shaker-like voltage-gated K+ channel, whose defections are linked to neuronal deficiency and childhood epilepsy. Despite the important role in the kinetic behavior of the channel, the inactivation remained hereby elusive. Here, we studied the Kv1.2 inactivation via a combined simulation/network theoretical approach that revealed two distinct pathways coupling the Voltage Sensor Domain and the Pore Domain to the Selectivity Filter. Additionally, we mutated some residues implicated in these paths and we explained microscopically their function in the inactivation mechanism by computing a contact map. Interestingly, some pathological residues shown to impair the inactivation lay on the paths. In summary, the presented results suggest two pathways as the possible molecular basis of the inactivation mechanism in the Kv1.2 channel. These pathways are consistent with earlier mutational studies and known mutations involved in neuronal channelopathies.

Engineering of a Spider Peptide via Conserved Structure-Function Traits Optimizes Sodium Channel Inhibition In Vitro and Anti-Nociception In Vivo

Venom peptides are potent and selective modulators of voltage-gated ion channels that regulate neuronal function both in health and in disease. We previously identified the spider venom peptide Tap1a from the Venezuelan tarantula Theraphosa apophysis that targeted multiple voltage-gated sodium and calcium channels in visceral pain pathways and inhibited visceral mechano-sensing neurons contributing to irritable bowel syndrome. In this work, alanine scanning and domain activity analysis revealed Tap1a inhibited sodium channels by binding with nanomolar affinity to the voltage-sensor domain II utilising conserved structure-function features characteristic of spider peptides belonging to family NaSpTx1. In order to speed up the development of optimized NaV-targeting peptides with greater inhibitory potency and enhanced in vivo activity, we tested the hypothesis that incorporating residues identified from other optimized NaSpTx1 peptides into Tap1a could also optimize its potency for NaVs. Applying this approach, we designed the peptides Tap1a-OPT1 and Tap1a-OPT2 exhibiting significant increased potency for NaV1.1, NaV1.2, NaV1.3, NaV1.6 and NaV1.7 involved in several neurological disorders including acute and chronic pain, motor neuron disease and epilepsy. Tap1a-OPT1 showed increased potency for the off-target NaV1.4, while this off-target activity was absent in Tap1a-OPT2. This enhanced potency arose through a slowed off-rate mechanism. Optimized inhibition of NaV channels observed in vitro translated in vivo, with reversal of nocifensive behaviours in a murine model of NaV-mediated pain also enhanced by Tap1a-OPT. Molecular docking studies suggested that improved interactions within loops 3 and 4, and C-terminal of Tap1a-OPT and the NaV channel voltage-sensor domain II were the main drivers of potency optimization. Overall, the rationally designed peptide Tap1a-OPT displayed new and refined structure-function features which are likely the major contributors to its enhanced bioactive properties observed in vivo. This work contributes to the rapid engineering and optimization of potent spider peptides multi-targeting NaV channels, and the research into novel drugs to treat neurological diseases.

Voltage-clamp fluorometry analysis of structural rearrangements of ATP-gated channel P2X2 upon hyperpolarization

Gating of the ATP-activated channel P2X2 has been shown to be dependent not only on [ATP] but also on membrane voltage, despite the absence of a canonical voltage-sensor domain. We aimed to investigate the structural rearrangements of rat P2X2 during ATP- and voltage-dependent gating, using a voltage-clamp fluorometry technique. We observed fast and linearly voltage-dependent fluorescence intensity (F) changes at Ala337 and Ile341 in the TM2 domain, which could be due to the electrochromic effect, reflecting the presence of a converged electric field. We also observed slow and voltage-dependent F changes at Ala337, which reflect structural rearrangements. Furthermore, we determined that the interaction between Ala337 in TM2 and Phe44 in TM1, which are in close proximity in the ATP-bound open state, is critical for activation. Taking these results together, we propose that the voltage dependence of the interaction within the converged electric field underlies the voltage-dependent gating.

Functional and Structural Characterization of ClC-1 and Nav1.4 Channels Resulting from CLCN1 and SCN4A Mutations Identified Alone and Coexisting in Myotonic Patients

Non-dystrophic myotonias have been linked to loss-of-function mutations in the ClC-1 chloride channel or gain-of-function mutations in the Nav1.4 sodium channel. Here, we describe a family with members diagnosed with Thomsen’s disease. One novel mutation (p.W322*) in CLCN1 and one undescribed mutation (p.R1463H) in SCN4A are segregating in this family. The CLCN1-p.W322* was also found in an unrelated family, in compound heterozygosity with the known CLCN1-p.G355R mutation. One reported mutation, SCN4A-p.T1313M, was found in a third family. Both CLCN1 mutations exhibited loss-of-function: CLCN1-p.W322* probably leads to a non-viable truncated protein; for CLCN1-p.G355R, we predict structural damage, triggering important steric clashes. The SCN4A-p.R1463H produced a positive shift in the steady-state inactivation increasing window currents and a faster recovery from inactivation. These gain-of-function effects are probably due to a disruption of interaction R1463-D1356, which destabilizes the voltage sensor domain (VSD) IV and increases the flexibility of the S4-S5 linker. Finally, modelling suggested that the p.T1313M induces a strong decrease in protein flexibility on the III-IV linker. This study demonstrates that CLCN1-p.W322* and SCN4A-p.R1463H mutations can act alone or in combination as inducers of myotonia. Their co-segregation highlights the necessity for carrying out deep genetic analysis to provide accurate genetic counseling and management of patients.

Voltage-clamp fluorometry analysis of structural rearrangements of ATP-gated channel P2X2 upon hyperpolarization

ABSTRACTThe gating of the ATP-activated channel P2X2 has been shown to be dependent not only on [ATP] but also on membrane voltage, despite the absence of a canonical voltage-sensor domain. We aimed to investigate the structural rearrangements of the rat P2X2 during ATP- and voltage-dependent gating by voltage-clamp fluorometry technique. We observed fast and linearly voltage-dependent fluorescence intensity (F) changes at Ala337 and Ile341 in the TM2 domain, which could be due to the electrochromic effect, reflecting the presence of a converged electric field here. We also observed slow and voltage-dependent F changes at Ala337, which reflect the structural rearrangements. Furthermore, we identified that the interaction between Ala337 in TM2 and Phe44 in TM1, located in close proximity in the ATP-bound open state, is critical for activation. Taken together, we propose that the voltage dependence of the interaction in the converged electric field underlies the voltage-dependent gating.

A PIP2 substitute mediates voltage sensor-pore coupling in KCNQ activation

KCNQ family K+ channels (KCNQ1-5) in the heart, nerve, epithelium and ear require phosphatidylinositol 4,5-bisphosphate (PIP2) for voltage dependent activation. While membrane lipids are known to regulate voltage sensor domain (VSD) activation and pore opening in voltage dependent gating, PIP2 was found to interact with KCNQ1 and mediate VSD-pore coupling. Here, we show that a compound CP1, identified in silico based on the structures of both KCNQ1 and PIP2, can substitute for PIP2 to mediate VSD-pore coupling. Both PIP2 and CP1 interact with residues amongst a cluster of amino acids critical for VSD-pore coupling. CP1 alters KCNQ channel function due to different interactions with KCNQ compared with PIP2. We also found that CP1 returned drug-induced action potential prolongation in ventricular myocytes to normal durations. These results reveal the structural basis of PIP2 regulation of KCNQ channels and indicate a potential approach for the development of anti-arrhythmic therapy.

Gain of channel function and modified gating properties in TRPM3 mutants causing intellectual disability and epilepsy

Developmental and epileptic encephalopathies (DEE) are a heterogeneous group of disorders characterized by epilepsy with comorbid intellectual disability. Recently, two de novo heterozygous mutations in the gene encoding TRPM3, a calcium permeable ion channel, were identified as the cause of DEE in eight probands, but the functional consequences of the mutations remained elusive. Here we demonstrate that both mutations (V990M and P1090Q) have distinct effects on TRPM3 gating, including increased basal activity, higher sensitivity to stimulation by the endogenous neurosteroid pregnenolone sulfate (PS) and heat, and altered response to ligand modulation. Most strikingly, the V990M mutation affected the gating of the non-canonical pore of TRPM3, resulting in large inward cation currents via the voltage sensor domain in response to PS stimulation. Taken together, these data indicate that the two DEE mutations in TRPM3 result in a profound gain of channel function, which may lie at the basis of epileptic activity and neurodevelopmental symptoms in the patients.