Abstract

Voltage-gated sodium channels initiate action potentials in prokaryotes and in many eukaryotic cells, including vertebrate nerve and muscle. Their activation is steeply voltage-dependent, but it is unclear how the voltage sensitivity is set or whether it can be broadly shifted to positive voltages. Here we show that the voltage dependence of activation (VA) of the ancestral bacterial sodium channel NaVAb can be progressively shifted from -118 mV to +35 mV in chimeras with increasing numbers of amino acid residues from the extracellular half of the voltage sensor of human NaV1.7 channels. In a minimal chimera in which only 32 residues were transferred, we analyzed the effects of six additional mutations of conserved amino acid residues singly, in pairs, and as triple mutations. The resulting chimeric mutants exhibited a broad range of voltage sensitivity from VA=-118 mV to VA=+120 mV. Three mutations (N48K, L112A, and M119V) shifted VA to +61 mV when substituted in NaVAb itself, and substitution of two additional Cys residues in the Cys-free background of NaVAb further shifted VA to +105 mV. In these mutants, measurement of gating currents revealed that the voltage dependence of gating charge movement (VQ) shifted to positive membrane potentials as much or more than VA, confirming that the gating charges are trapped in their resting positions by these VA-shifting mutations. Our results demonstrate broadband shifting of VA and VQ of a sodium channel across a range of 240 mV and provide a toolbox of methods and constructs to analyze sodium channel structure and function in the resting state at 0 mV and in activated states at positive membrane potentials. GRAPHICAL ABSTRACTThe complete range of broadband tuning of voltage-dependent activation of a sodium channel. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/392571v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@147c265org.highwire.dtl.DTLVardef@2b8b15org.highwire.dtl.DTLVardef@b84b48org.highwire.dtl.DTLVardef@1569072_HPS_FORMAT_FIGEXP M_FIG C_FIG