Figure 1.
Figure 1. NaVSp1-P pore-domain adopts a conductive conformation. (A) A schematic representation of the full-length NaVSp1 with the VSD and the PD marked. Only two subunits are shown for clarity. An exemplar recording of NaVSp1 whole-cell Na+ currents. The activation protocol consisted of 500-ms depolarization from −80 to +40 mV in 5-mV increments from a holding potential of −120 mV with a sweep-to-sweep interval of 4 s. The inset shows a typical response to 0-mV depolarization pulse, where the steady-state open probability is less than 5% of the peak value (left). NaVSp1 currents elicited by pulse-protocol (shown above). At the end of a 0-mV prepulse, the test pulse to 10 mV evokes minimal inward current, indicating that the channels are inactivated at the end of the prepulse (right). (B) Single-channel recordings of NaVSp1-P reconstituted in liposomes (a schematic of NaVSp1-P shown above) show functionally active channels that stochastically open and close. The trace within the gray box is shown in an expanded scale. C and O denote the closed and open states. The dotted line marks the zero-current level. (C) Two diagonal subunits of closed-gate NaVAb crystal structure (PDB ID 3RVY) with the position at the bundle crossing (I215) shown as black spheres. The boxed regions highlight the activation gate and the selectivity filter region in the channel. Spin-normalized CW-EPR spectra of NaVSp1-P I215C in asolectin membrane (bottom). Background-corrected Q-band DEER echo intensity is plotted against evolution time for I215R1 in DM (middle panel) and fit using model-free Tikhonov regularization. The Tikhonov L-curve is shown in the inset. The corresponding interspin distance distribution with regularization parameter (α = 100) is shown in the right panel, with two distributions corresponding to the distances between the adjacent and nonadjacent subunits. For the position I215C, interspin distance distributions were simulated with NaV structures (PDB ID 3RVY for closed, orange; and PDB ID 3ZJZ for open, green) based on analysis of spin label rotamers using the MMM software package (Polyhach et al., 2007, 2011). Rotamer library calculations were conducted at 83 K.

NaVSp1-P pore-domain adopts a conductive conformation. (A) A schematic representation of the full-length NaVSp1 with the VSD and the PD marked. Only two subunits are shown for clarity. An exemplar recording of NaVSp1 whole-cell Na+ currents. The activation protocol consisted of 500-ms depolarization from −80 to +40 mV in 5-mV increments from a holding potential of −120 mV with a sweep-to-sweep interval of 4 s. The inset shows a typical response to 0-mV depolarization pulse, where the steady-state open probability is less than 5% of the peak value (left). NaVSp1 currents elicited by pulse-protocol (shown above). At the end of a 0-mV prepulse, the test pulse to 10 mV evokes minimal inward current, indicating that the channels are inactivated at the end of the prepulse (right). (B) Single-channel recordings of NaVSp1-P reconstituted in liposomes (a schematic of NaVSp1-P shown above) show functionally active channels that stochastically open and close. The trace within the gray box is shown in an expanded scale. C and O denote the closed and open states. The dotted line marks the zero-current level. (C) Two diagonal subunits of closed-gate NaVAb crystal structure (PDB ID 3RVY) with the position at the bundle crossing (I215) shown as black spheres. The boxed regions highlight the activation gate and the selectivity filter region in the channel. Spin-normalized CW-EPR spectra of NaVSp1-P I215C in asolectin membrane (bottom). Background-corrected Q-band DEER echo intensity is plotted against evolution time for I215R1 in DM (middle panel) and fit using model-free Tikhonov regularization. The Tikhonov L-curve is shown in the inset. The corresponding interspin distance distribution with regularization parameter (α = 100) is shown in the right panel, with two distributions corresponding to the distances between the adjacent and nonadjacent subunits. For the position I215C, interspin distance distributions were simulated with NaV structures (PDB ID 3RVY for closed, orange; and PDB ID 3ZJZ for open, green) based on analysis of spin label rotamers using the MMM software package (Polyhach et al., 2007, 2011). Rotamer library calculations were conducted at 83 K.

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