Patch Trauma-Induced Kinetic Changes in Nav1.4 Channels (Oocyte Patches): Gating in Both a Nav1.4-Nav1.5 Chimera Insusceptible to β1 Modulation and a Nav1.4 Mutant Incapable of Binding G-Ankyrin is Irreversibly Modulated by Membrane Stretch

Oocytes lack endogenous Nav-modulating β subunits. Nav1.4α (unlike Nav1.5α) pore subunits expressed in oocytes exhibit anomalously slow, right-shifted voltage-dependent gating but co-expression with β1 subunits results in normal (i.e. more rapid, more left-shifted) Nav1.4 kinetics. For unknown reasons, membrane stretch due to pipette aspiration in cell-attached oocyte patches has the same effect on Nav1.4 α kinetics as co-expression of α with β1, namely, acceleration/left-shift of the activation and availability processes. This also occurs with Nav1.6 (Wang et al 2009 Am J Physiol 297:C823) where the nature of the kinetic changes in Nav1.6 suggests trauma-induced changes in bilayer mechanics as a plausible explanation. However, mechanical disruption between Navα and various protein “targets” might also be the explanation. Makita et al 1996 (J Neurosci 16:7117) generated a Nav1.4-Nav1.5 chimera in which the re-entrant S5-S6 “pore loops” of Nav1.4 domains 1 and 4 were substituted with the corresponding Nav1.5 pore loops. This rendered the chimera incapable of modulation by β1 subunits. Here we show that slow component activity in this chimera was irreversibly accelerated and left-shifted by membrane stretch. We also tested a rat Nav1.4α mutant in which the cytoplasmic loop 2 amino acids responsible for binding to G-ankyrin have been deleted. In this Nav1.4α mutant too, patch stretch irreversibly accelerated/left-shifted the anomalously slow activity of the channel. These findings rule out the possibility that the extracellular β1 interacting domains of Nav1.4α or the intracellular G-ankyrin interacting domain of Nav1.4α are required for the irreversible membrane trauma-induced acceleration/left-shift of this channel.