Eslam Elhanafy

Voltage Sensor Domains (VSDs) are integral and common regions within voltage-gated ion channels that play a crucial role in cellular signaling and excitability. It has four transmembrane segments (S1-S4) per domain. The transmembrane segment S4 contains positively charged amino acid residues at every third position, which serve as the channel's gating charges. Over the last decade, it has been shown that these gating charges are the target of many pathological mutations. This study investigates three gating charge mutations in Domain I of the NaV1.2 channel (R220G, R223I, R223Q) identified in patients with distinct clinical phenotypes. The three mutations showed differential gating-pore current amplitudes with distinct voltage dependence. To elucidate the molecular basis for the differential Igp observed among these mutants, we performed microsecond-scale molecular dynamics (MD) simulations and a comprehensive analysis of the state-dependent interaction network within VSDI across WT and mutant conformational states. The results showed that cation-π interactions between the S4 gating charges and the hydrophobic residue in S2 are the main forces that prevent VSD from leaking gating-pore current. Hydrophobic interactions represent the second line of defense against VSD leaking, preventing gating-pore formation by maintaining structural integrity across different conformations. Salt-bridge interactions and hydrogen bonds are important stabilizing forces; however, within the voltage-sensing domain, their contribution appears to be less significant than that of cation–π and hydrophobic interactions. Predicting and identifying gating pore currents as a potential shared pathophysiology across diverse clinical presentations may guide the design of targeted therapeutic strategies.

Voltage-gated sodium (Nav) channels are pivotal for cellular signaling, and mutations in Nav channels can lead to excitability disorders in cardiac, muscular, and neural tissues. A major cluster of pathological mutations localizes in the voltage-sensing domains (VSDs), resulting in either gain-of-function, loss-of-function effects, or both. However, the mechanism behind this functional diversity of mutations at equivalent positions remains elusive. Through hotspot analysis, we identified three gating charges (R1, R2, and R3) as major mutational hotspots in VSDs. The same amino acid substitutions at equivalent gating-charge positions in VSDI and VSDII of the cardiac sodium channel Nav1.5 show differential gating property impacts in electrophysiology measurements. We conducted molecular dynamics (MD) simulations on wild-type channels and six mutants to elucidate the structural basis of their differential impacts. Our 120-µs MD simulations with applied external electric fields captured VSD state transitions and revealed the differential structural dynamics between equivalent R-to-Q mutants. Notably, we observed transient leaky conformations in some mutants during structural transitions, offering a detailed structural explanation for gating-pore currents. Our salt-bridge network analysis uncovered VSD-specific and state-dependent interactions among gating charges, countercharges, and lipids. This detailed analysis revealed how mutations disrupt critical electrostatic interactions, thereby altering VSD permeability and modulating gating properties. By demonstrating the crucial importance of considering the specific structural context of each mutation, our study advances our understanding of structure–function relationships in Nav channels. Our work establishes a robust framework for future investigations into the molecular basis of ion channel–related disorders.

Voltage-gated sodium (Na _v ) channels are pivotal for cellular signaling and mutations in Na _v channels can lead to excitability disorders in cardiac, muscular, and neural tissues. A major cluster of pathological mutations localizes in the voltage-sensing domains (VSDs), resulting in either gain-of-function (GoF), loss-of-function (LoF) effects, or both. However, the mechanism behind this functional divergence of mutations at equivalent positions remains elusive. Through hotspot analysis, we identified three gating charges (R1, R2, and R3) as major mutational hotspots in VSDs. The same amino-acid substitutions at equivalent gating-charge positions in VSD _I and VSD _II of the cardiac sodium channel Na _v 1.5 show differential gating-property impacts in electrophysiology measurements. We conducted 120 µs molecular dynamics (MD) simulations on wild-type and six mutants to elucidate the structural basis of their differential impacts. Our μs-scale MD simulations with applied external electric fields captured VSD state transitions and revealed the differential structural dynamics between equivalent R-to-Q mutants. Notably, we observed transient leaky conformations in some mutants during structural transitions, offering a detailed structural explanation for gating-pore currents. Our salt-bridge network analysis uncovered VSD-specific and state-dependent interactions among gating charges, countercharges, and lipids. This detailed analysis elucidated how mutations disrupt critical electrostatic interactions, thereby altering VSD permeability and modulating gating properties. By demonstrating the crucial importance of considering the specific structural context of each mutation, our study represents a significant leap forward in understanding structure- function relationships in Na _v channels. Our work establishes a robust framework for future investigations into the molecular basis of ion channel-related disorders.

Thousands of voltage-gated sodium (Nav) channel variants contribute to a variety of disorders, including epilepsy, autism, cardiac arrhythmia, and pain disorders. Yet variant effects of more mutations remain unclear. The conventional gain-of-function (GoF) or loss-of-function (LoF) classifications is frequently employed to interpret of variant effects on function and guide precision therapy for sodium channelopathies. Our study challenges this binary classification by analyzing 525 mutations associated with 34 diseases across 366 electrophysiology studies, revealing that diseases with similar phenotypic effects can stem from unique molecular mechanisms. Our results show a high biophysical agreement (86%) between homologous disease-associated variants in different Na _v genes, significantly surpassing the 60% phenotype (GoF _o /LoF _o ) agreement among homologous mutants, suggesting the need for more nuanced disease categorization and treatment based on specific gating-property changes. Using UniProt data, we mapped over 2,400 disease-associated missense variants across nine human Nav channels and identified three clusters of mutation hotspots. Our findings indicate that mutations near the selectivity filter generally diminish the maximal current amplitude, while those in the fast inactivation region lean towards a depolarizing shift in half-inactivation voltage in steady-state activation, and mutations in the activation gate commonly enhance persistent current. In contrast to mutations in the PD, those within the VSD exhibit diverse impacts and subtle preferences on channel activity. This study shows great potential to enhance prediction accuracy for variant effects based on the structural context, laying the groundwork for targeted drug design in precision medicine.