COEVOLUTION AND CHARACTERIZATION OF MUTANT VOLTAGE- DEPENDENT SODIUM CHANNELS

Voltage-dependent sodium channels (Nav) are crucial for generating and propagating action potentials in cardiac cells. The specific arrangement and amino acid composition of these channels are closely tied to their function, meaning that mutations or substitutions in certain residues can lead to channelopathies, which may disrupt heart rhythm. In this study, we focused on the Nav 1.5 channel, the primary variant in mammalian hearts, using PFstats software and the Residue Coevolution Network Decomposition method to identify key amino acid residues that coevolve and play a significant role in channel inactivation. We conducted coevolutionary analyses and reconstructed the evolutionary history of the IFMT motif (Isoleucine-Phenylalanine-Methionine-Threonine), a critical component in the rapid inactivation of sodium channels, using protein sequences and the MEGA XI software. Additionally, we selected three amino acid residues that coevolve with the IFMT motif to evaluate the mutant phenotypes of Nav 1.5 using the Patch-clamp technique. Our findings reveal a connection between the fast inactivation gate of the sodium channel and 27 amino acids modulated by either the IFMT or LFLT motifs. Phylogenetic reconstruction of Nav 1.5 sequences from mammals, reptiles, and birds showed that mammals conserve the IFMT motif, while some reptiles exhibit modifications forming the LFLT motif (Leucine-Phenylalanine-Leucine-Threonine). Interestingly, some bird species possess both IFMT and LFLT motifs. Structural analysis of the loops formed by the IFMT and LFLT motifs revealed a shortening of the alpha helix connecting the loop formed by LFLT and the fourth transmembrane domain. Further evolutionary and structural analyses aim to elucidate how these structural differences impact the fast inactivation functionality of the sodium channel. Additionally, evaluating selected mutants using the Patch-clamp technique will help establish the modulatory relationship between coevolving residues and sodium channel fast inactivation.