Gating of the bacterial sodium channel, NaChBac: voltage-dependent charge movement and gating currents

Abstract

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 micros 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.

Figure 1.

NaChBac gating current characteristics. (A) Original gating current traces elicited by a family of 10-ms depolarizations from a −150 mV HP to voltages ranging from −120 to 30 mV in 10-mV steps. Typical traces for several test pulse potentials are shown. (B) Charge–voltage (Q–V) relationship (open circles) from an HP of −150 mV, compared with Q–V from an HP of 0 mV (open squares). Curves are the fits to single Boltzmann functions of the form: I(V) = A2 + (A1 − A2)/(1 + exp(−(V − V _1/2)/k)), where A1 and A2 are amplitudes, V is the voltage, V _1/2 is the half-maximal voltage, and k is the slope factor. The fitted parameters were: V _1/2 = −54 ± 2 mV, k = 12.3 ± 1.6, n = 4 (HP = −150 mV) vs. V _1/2 = −113 ± 5 mV, k = 19.1 ± 3.3, n = 3 (HP = 0 mV), where n is the number of cells.

Figure 2.

NaChBac ionic current characteristics. (A) Representative I_Na traces elicited by a family of 10-ms depolarizations from a −150-mV HP to voltages ranging from −120 to 80 mV in 10-mV steps. (B) Normalized current–voltage (I–V) relationships. Data were fit to a sigmoid function with V_1/2 = −36 ± 1 mV and k = −5.6 ± 0.1, n = 4. (C) Instantaneous I–V curve obtained from normalized tail current amplitudes (n = 5) at the voltages plotted, following a 10-ms prepulse to −10 mV. HP was −120 mV.

Figure 3.

Cole-Moore shift. (A) I_Na traces elicited by a test pulse to −10 mV following a 100-ms preconditioning pulse to various potentials. For better resolution, recordings were done at the room temperature (∼21°C). HP was −150 mV. The circle indicates the region with expanded time resolution shown below. (B) Time shift to the half-maximal ionic current for indicated voltages (n = 5).

Figure 4.

Charge movement and ionic conductance. (A) Q–V and G–V for an HP of −150 mV. Q–V is shifted by 22 mV toward more negative potentials and has less voltage dependence relative to the G–V. Both curves are the fits to Boltzmann functions with fit parameters: V_1/2 = −54 ± 2 mV, k = 12.3 ± 1.6 (Q–V, n = 4) vs. V_1/2 = −32 ± 2 mV, k = 8.0 ± 0.4 (G–V, n = 5), where V_1/2 is the midpoint potential of the curve, k is the slope factor, and n is the number of cells. (B) Single exponential time constants of I_{g ON} decay (n = 6) and time to half-maximal ionic current as a function of voltage (n = 4).

Figure 5.

Kinetic simulations. (A) Sequential simplified model used for kinetic simulations. C₁–C₄ are four closed states, O is the open state, I is the inactivated state, and α₁, α₂, β₁, and β₂ are voltage-dependent transition rates between the states, as shown in the model. For simplicity, all transition rates in the activation pathway, C4 through O, were assumed to be equal. (B) Representative ionic current traces elicited by a family of 500-ms depolarizations from a −150-mV HP to voltages ranging from −40 to 0 mV in 10-mV steps, simultaneously fitted to the sequential model of NaChBac gating shown in A. Superimposed on the traces are the fitted curves. Inset, same as part B with expanded time scale. (C) Fit of predicted time course of gating currents compared with experimental data fits.