Divergence in Domain IV of an Electric Fish NaV Channel Tunes its Fast Inactivation to Support Rapid Firing Rates by Electro-Motorneurons

Abstract

In some neuronal cell types, persistent or resurgent current through voltage-gated sodium channels enables the regular firing of “spontaneous” action potentials from a few to a hundred Hz. However, the neurons with the fastest spontaneous firing rates known are found in the nocturnal ghost knifefish (family apteronotidae), which accomplish active electrolocation via a unique neuronal electrical organ that spontaneously fires action potentials at rates exceeding 1000 Hz. Voltage-clamping of electro-motorneurons from apteronotidae revealed voltage-gated sodium currents with incomplete fast inactivation (i.e., the existence of a persistent sodium current). Unexpectedly, RT-PCR revealed that Nav 1.4b is the dominant Nav isoform in the apteronotid spinal cord, and it is expressed here nearly exclusively. We found that this gene contains five apteronotid-specific substitutions in the S4-S5 cytoplasmic linker of Domain IV, a region that has been implicated in the process of fast inactivation of mammalian voltage gated sodium channels. Using two electrode voltage clamp electrophysiology, we assayed the effects of making combinations of these substitutions in the human cardiac sodium channel Nav 1.5 (R1644W, L1647W, M1651R, I1660F, G1661S). Interestingly, when all five apteronotid substitutions are incorporated into DIV S4-5 of Nav 1.5, a persistent sodium current is observed that is qualitatively similar to that of the apterotontid Nav 1.4b in native cells. In addition, the effects observed in some partial substitutions (for example, loss of voltage dependence of inactivation in R1644W/M1651R-Nav 1.5), were apparently lost in the context of the quintuple-mutant. Taken together, these results suggest that the evolution of rapid electrical organ discharge in knifefish was driven by the tissue-specific expression of and sequence divergence within the voltage gated sodium channel Nav 1.4b. Moreover, they help define the structural requirements within the DIV S4-S5 linker for normal inactivation in mammalian sodium channels.