Multiple gating processes associated with the distal end of the S6 segment of domain II in the Nav channels

Figures

• 

  Figure 1. The Leu site mutation in the distal end of the DII S6 segment hyperpolarized the activation of Nav channel.A, the human Nav1.9 structure was predicted by the AlphaFold2 (39) and the structure is domain colored. Three perpendicular views are shown in cartoon, side view (upper left), bottom view (upper right) and up view (lower left). Inset: Intracellular gate (lower right). The L811 site is colored green. B, sequences alignments corresponding to the DII S6 segment of human Nav channels and rat Nav1.4, the L811 site located the end of DII S6 segment of Nav1.9, it is highly conserved in Nav channels. C, representative current traces in ND7/23 cells transfected with Nav1.9 WT (n = 10), L811P (n = 14), L811A (n = 8), L811C (n = 5), and L811N (n = 10), respectively. Scale bar 1 nA, 20 ms. The inset shows the current elicited protocol. D, the current density-voltage relationships of Nav1.9 WT and L811 mutations for data in (C). E, steady-state activation curves fit with a Boltzmann function (Equation 3) (n = 5–14). F, scatter dot plot showing the midpoint of activation potential for WT and L811 mutations (one-way ANOVA with post hoc analysis using Dunnett's multiple comparisons test, n…

• 

  Figure 2. The L811 mutations make the docking of the IFMT motif with the binding site unstable. A, representative normalized current traces showing fast inactivation kinetics of Nav1.9 L811 mutants compared with Nav1.9 WT (n = 5–14). B, L811 mutants significantly enhance the persistent current of the channel compared with Nav1.9 WT (n = 10 for WT, n = 14 for L811P, n = 8 for L811A, n = 5 for L811C, and n = 10 for L811N) (two-way ANOVA followed by Tukey’s multiple comparisons test). C, steady-state inactivation of Nav1.9 WT and L811 mutants in ND7/23 cells were measured with a series of 500-ms prepulse (−140 mV or −120 mV to −10 mV in 10-mV increments), followed by a 50-ms depolarization to test potential (−40 mV for WT, L811A and L811N, −60 mV for L811P) to assess the available non-inactivated channels. The ND7/23 cells held at −120 mV (WT, L811A L811C and L811N) or −140 mV (L811P). Data points were well fit with a Boltzmann function (Equation 3) (n = 8 for WT, n = 13 for L811P, n = 10 for L811A, and n = 12 for L811N). D, scatter dot plot showing the midpoint of inactivation potential for WT and L811 mutations (one-way ANOVA with post hoc analysis using the Dunnett's multiple comparisons…

• 

  Figure 3. L811P accelerates the slow inactivation.A, the steady-state slow inactivation curves of Nav1.9 WT and L811 mutations, data points for slow inactivation kinetics were well fitted with the Boltzmann equation (Equation 3, left). Scatter dot plot showing the midpoint of slow inactivation potential for WT and L811 mutations (right) (n = 19 for WT, n = 12 for L811P, n = 7 for L811A, and n = 6 for L811N) (one-way ANOVA with post hoc analysis using the Dunnett's multiple comparisons test). The slow inactivation was measured with a series of 30-s prepulses (−140 or −120 mV to −50 mV in 10-mV increments), followed by a 100-ms pulse to the holding potential to remove fast-inactivation, and then depolarization to −60 mV (L811P) or −40 mV for a duration of 50-ms (WT, L811A and L811N) to assess the available non-inactivated channels. The ND7/23 cells held at −120 mV (WT, L811A and L811N) or −140 mV (L811P). B, the steady-state slow inactivation curves are shifted −25 mV by Nav1.4 L796P (left). Scatter dot plot showing the midpoint of slow inactivation potential for WT and L796P (right) (n = 11 for WT and n = 10 for L796P) (two-tailed t test). The inset shows the current elicited protocol, and…

• 

  Figure 4. The Leu site mutation in the distal end of the DII S6 segment accelerates the use-dependent inactivation of Nav channels.A and B, Upper, representative Na+ currents elicited in ND7/23 cells expressing the WT (black), L811P (red) or L811A (orange) hNav1.9 channel by a 0.2 Hz (A, n = 4 for WT, n = 4 for L811P and n = 5 for L811A) and 1 Hz (B, n = 4 for WT, n = 4 for L811P and n = 5 for L811A) pulse train. The cells, held at −120 mV (WT and L811A) or −140 mV (L811P), were depolarized to −40 mV (WT) or −60 mV (L811P and L811A) for 50-ms repetitive pulses applied at 0.2 Hz (A) or 1 Hz (B). Scale bar 1 nA, 20 ms. Lower, the current amplitudes are normalized to those elicited by the first pulse. C and D, Upper, representative Na+ currents elicited in HEK293T cells expressing the WT (black) or L796P (red) Nav1.4 channel by a 0.2 Hz (C, n = 7 for WT, n = 3 for L796P) and 1 Hz (D, n = 7 for WT, n = 3 for L796P) pulse train. The cells, held at −90 mV were depolarized to 0 mV) for 50-ms repetitive pulses applied at 0.2 Hz (C) or 1 Hz (D). Scale bar 1 nA, 20 ms. Lower, the current amplitudes are normalized to those elicited by the first pulse. Data are presented as mean ± SD.

• 

  Figure 5. L811P alters the permeabilities of channels to K+, Cs+, and Ca2+. A–C, from top to bottom, the left panels show representative current traces from WT and L811P channels in KCl- (A), CsCl- (B), and CaCl2-based (C) extracellular solutions in CHO-K1 cells, the inset shows the current evoked protocol. For WT, the holding potential is −120 mV. For L811P, the holding potential is −140 mV. The middle panels show the I-V curves for WT and L811P in the NaCl-based external solution before substituting K+, Cs+ or Ca2+. The right panels show I-V relationships for WT and L811P channels in KCl- (A, n = 7 for WT, n = 11 for L811P), CsCl- (B, n = 7 for WT, n = 7 for L811P), and CaCl2-based (C, n = 9 for WT, n = 8 for L811P) solutions. Data are presented as mean ± SD.