Gating of the Bacterial Sodium Channel, NaChBac: Voltage-dependent Charge Movement and Gating Currents

The bacterial sodium channel, NaChBac, from Bacillus halodurans provides an excellent model to study structure–function relationships of voltage-gated ion channels. It can be expressed in mammalian cells for functional studies as well as in bacterial cultures as starting material for protein purification for fine biochemical and biophysical studies. Macroscopic functional properties of NaChBac have been described previously (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001. Science . 294:2372–2375). In this study, we report gating current properties of NaChBac expressed in COS-1 cells. Upon depolarization of the membrane, gating currents appeared as upward inflections preceding the ionic currents. Gating currents were detectable at 90 mV while holding at 150 mV. Charge–voltage (Q–V) curves showed sigmoidal dependence on voltage with gating charge saturating at 10 mV. Charge movement was shifted by 22 mV relative to the conductance–voltage curve, indicating the presence of more than one closed state. Consistent with this was the Cole-Moore shift of 533 s observed for a change in preconditioning voltage from 160 to 80 mV. The total gating charge was estimated to be 16 elementary charges per channel. Charge immobilization caused by prolonged depolarization was also observed; Q–V curves were shifted by approximately 60 mV to hyperpolarized potentials when cells were held at 0 mV. The kinetic properties of NaChBac were simulated by simultaneous fit of sodium currents at various voltages to a sequential kinetic model. Gating current kinetics predicted from ionic current experiments resembled the experimental data, indicating that gating currents are coupled to activation of NaChBac and confirming the assertion that this channel undergoes several transitions between closed states before channel opening. The results indicate that NaChBac has several closed states with voltage-dependent transitions between them realized by translocation of gating charge that causes activation of the channel. key words: sodium channel • gating charge • bacterial channels • C-type inactivation • Cole-Moore shift I N T R O D U C T I O N Voltage-gated ion channels are specialized proteins embedded in the cell membrane whose function is controlled by the electrical potential gradient across the membrane and that posses ion-conducting pores that are selective to a particular ion. Despite differences in their physiological functions, the general channel structure and the gating mechanisms show high evolutionary conservation among different channels that allows inferences on structure–function relationships from one channel type to the next. A new group of voltage-gated ion channels is emerging from the analysis of bacteria genomes. Recently, a voltage-gated Na channel has been reported (Ren et al., 2001), the NaChBac obtained from Bacillus halodurans . Intriguingly, this channel reveals structural and functional features different from all other voltage-gated Na channels described. First, it is built of only one domain containing six transmembrane segments connected by extraand intracellular loops. Thus, it resembles the structure of most voltage-gated K channels, rather than that of voltage-gated Na or Ca 2 channels where a large pore-forming -subunit consists of four six-transmembrane pseudodomains. Similar to K channels, NaChBac monomers presumably assemble as tetramers to form functional channels. Second, despite its high selectivity to Na , NaChBac has a pore structure similar to that of voltage-gated Ca 2 channels (Durell and Guy, 2001; Ren et al., 2001; Yue et al., 2002). Third, NaChBac pharmacology resembles that of Ca 2 channels rather than that of Na channels, being sensitive to block by divalent cations and agents like Nifedipine, and mostly insensitive to tetrodotoxin (Ren et al., 2001). Finally, NaChBac Na current kinetics, including activation, inactivation, and recovery from inactivation are 10–100 Address correspondence to Ana M. Correa, Department of Anesthesiology, David Geffen School of Medicine at University of California, Los Angeles, BH-509A, CHS Box 957115, Los Angeles, CA 900957115. Fax: (310) 794-9612; email: [email protected] A. Kuzmenkin was on leave from the Department of Applied Physiology, University of Ulm, D-89069 Ulm, Germany, his current address. Abbreviations used in this paper: HP, holding potential; MS, methane sulfonate; NMG, N -methyld -glucamine. on N ovem er 7, 2016 D ow nladed fom Published September 13, 2004