Role of N- and C- termini in inactivation of sodium channel in weakly electric fish

The weakly electric fish Sternopygus macrurus emits an electric organ discharge (EOD) composed of a series of pulses. The EOD pulse is mainly shaped by sodium currents. There are two sodium channel α subunits orthologs of the mammalian Nav1.4 expressed in the EO of Sternopygus. Previous studies identified a novel splice variant of the Nav1.4b (Nav1.4bL), in which an extra 51-amino acid occurs in the N terminal end. Nav1.4bL currents inactivate and recover from inactivation significantly faster than Nav1.4bS. The voltage-dependence of steady-state inactivation of smNav1.4bL shifts to hyperpolarized potential. Structural analysis predicts an α-helix in the middle of the extended N terminus. Removal of a proline right after the α-helix significantly slows down current decay but has no effect on channel recovery from inactivation, suggesting inactivation and recovery have independent mechanism. Mutagenesis analysis of the extended N terminus showed that the short helical region, especially the positive charges in the helix, is an important determinant for channel voltage-dependence of steady-state inactivation. However, other residues outside the helical region are required for regulation of fast inactivation and recovery form inactivation. Functional and structural analysis provides evidence for the importance of the C terminus in fish Nav1.4b channel properties. Chimera in which the C terminus of smNav1.4bS was substituted by the human Nav1.4 C terminus, shows an 11 mV positive shift in voltage-dependence of activation and a -16 mV negative shift in inactivation. Deletion of the distal half of smNav1.4bS negatively shifted voltage-dependence of inactivation and significantly accelerated channel recovery from inactivation. In the deletion mutant, the regulation by the N segment is missing. Substitution of the C terminus mutant retains wild type channel inactivation and recovery properties and can be regulated by N segment again. My study provides evidence that the extended N terminus of smNav1.4bL binds the distal part of C terminal tail to modulate channel inactivation properties. This is the first time to show the distal C terminus is involved in channel recovery from inactivation. Studies in the fish sodium channel properties provide useful information to understand function and structure of voltage-gated sodium channels.