Mutagenesis of the NaChBac sodium channel discloses a functional role for a conserved S6 asparagine

Figures

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  Fig. 1. Sequence alignment of S6 segments. NaChBac, NavAb, NavMs, and NavRh are bacterial sodium channels and Nav1.1, Cav1.1 and TRP are human channels. The conserved S6 asparagine is shaded in black. Residue numbers are listed for the predicted start and end of the NaChBac S6 segment (depicted as a cartoon cylinder over the sequence). The sequence alignment was performed using T-COFFEE (http://www.ebi.ac.uk/tools/msa/tcoffee/)

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  Fig. 2. Analysis of activation and inactivation kinetics of NaChBac WT and mutant. a Representative current traces (lower panel) for activation of NaChBac-WT (left) and N225D mutant (right) expressed in HEK293t cells in response to the voltage protocol shown (top panel). Pulses for eliciting currents of N225D were shorter in order to prevent rundown. b Normalized current evoked by a series of test pulses to +50 mV at 40-s intervals for NaChBac-WT (300-ms test pulse length, squares, n = 7) and N225D mutant (50-ms test pulse length, circles, n = 10). No apparent run down is obvious. c Conductance-voltage relation of NaChBac-WT (squares) and N225D mutant (circles). The conductance was calculated from peak currents evoked by the protocol shown in a, normalized and plotted against the test pulse voltage. Data were fitted to a Boltzmann equation (solid line for WT, dotted line for N225D). d Voltage-dependence of steady-state inactivation of NaChBac-WT (squares) and N225D mutant (circles). Values were obtained by a test pulse to +50 mV following a conditioning prepulse incrementing in 5-mV steps (see inset). Peak currents were normalized to the peak current at the test pulse and plotted against…

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  Fig. 3. Thermal denaturation studies of NaChBac using SRCD spectroscopy. SRCD spectra of a WT and b N225D NaChBac measured over the temperature range of 20 °C (black solid lines) to 85 °C (black dotted lines) in 5 °C increments. Grey spectra are intermediate temperatures. Insets show the chemical structure of each 225 residue side chain. c Thermal denaturation curves of WT (solid line) and N225D (dashed line) derived from plotting the signals at 193 nm of spectra (a, b) versus temperature. Error bars represent 1 standard deviation between two repeats. d Fraction of first basis spectra as a function of temperature obtained by carrying out singular value deconvolution analyses (32) on the full spectra. The data are fitted to a Boltzmann function

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  Fig. 4. Hydrogen bonds involving S6 asparagines in the crystal structures of NavRh (PDB ID 4DXW) and NavAb (PDB ID 4EKW) in inactivated conformations. a Transmembrane view of the NavRh pore module shown in ribbon format with S4-S5 linkers colored yellow, S5 helices purple, P-loops orange, S6 helices beige. The conserved S6 asparagines are in spacefill. b–d Asparagines are shown as sticks and hydrogen bonds as green dashes for b NavRh chain B N213, c NavRh chain C N213, and d NavAB chain A N211