Fig 1.
Expression and biophysical properties of human Nav1.9 stably expressed in HEK-293 cells.
(A) Comparison of peak current density (pA/pF) of inward currents elicited by 40 ms voltage steps from -140 mV to -40 mV in HEK-293 cells stably expressing Nav1.9 alone or coexpressed with β1 and β2 subunits in presence and absence of 500 μM GTP-γ-S in intracellular solution. (B) Representative family current traces recorded in HEK-293 cells expressing Nav1.9 + β1/β2 recorded with 500 μM GTP-γ-S in pipette following a series of incremental voltage steps from –140 mV to +40 mV. (C) Inward current traces elicited in Nav1.9 + β1/β2 by 40 ms voltage steps from -140 mV to -40 mV in extracellular buffer containing 135 mM Na+ in absence or presence 10 μM tetrodotoxin (TTX) or in an extracellular buffer where all the sodium was replaced by choline. (D) Current density voltage relationship recorded in HEK-293 cells stably expressing Nav1.9 + β1/β2 + 500 μM GTP-γ-S (data are mean ± SEM from n = 13 observations). (E) Voltage dependence of activation and inactivation of Nav1.9 + β1/β2 recorded in the presence and absence of 500 μM GTP-γ-S following incremental depolarizing 100 ms conditioning voltage steps from -140 mV followed by a 40 ms test pulse to -40 mV. Data was fitted to a Boltzmann equation with parameters shown in Table 2. (F) Time course of recovery inactivation induced by either a 100 ms (circle) or 5 s (triangle) depolarizing voltage step to 0 mV. Recovery was assessed by applying a 40 ms test pulse to -40 mV after variable periods at -140 mV. Data was fit with a two phase exponential equation with fitted fast and slow time constants shown in Tables 3 and 4.
Table 1.
Current Density of HEK-293 hNav1.9 Stable Cell Lines.
Table 2.
Voltage Dependence of Activation and Inactivation.
Table 3.
Time Constants for Recovery from Inactivation Induced by 100 ms Prepulse to 0 mV.
Table 4.
Time Constants for Recovery from Inactivation Induced by 5 s Prepulse to 0 mV.
Fig 2.
Pharmacological modulation of human Nav1.9.
(A) Magnitude of Nav1.9 inactivation at different holding potentials. Percent inactivation determined from decline in test pulse current amplitude applied every 20s from test holding potential to -40 mV (values are mean ± SEM for 11–12 observations). (B) Comparison of concentration dependence of Nav1.9 inhibition by tetracaine at different holding potentials. (C) Representative current traces recorded in HEK-hNav1.9 cell line in the presence and absence of 3 μM TC-N 1752 (structure shown) during a 40 ms voltage step from -120 mV to -40 mV. (D) Diary plot of Nav1.9 current amplitude recorded using 40 ms voltage steps to -40 mV applied at 0.05 Hz, initially from a holding potential of -140 mV, then at -120 mV to induce ~25% inactivation. After current amplitude was stable at -120 mV, test agent was applied. Washout of test agent at holding potential of -140 mV until current amplitude became stable and then holding potential reset to -120 mV to allow inhibition to be determined by dividing current amplitude in presence of compound by interpolated current amplitude (see dotted line) between prior to compound addition and after washout. (E) Concentration response relationships for inhibition of Nav1.9 currents by known Nav channel inhibitors TC-N 1752, tetracaine, mexiletine and benzocaine. IC50s for these agents are plotted in (F) (values are summarized in Table 5) and are compared to IC50s for inhibition of Nav1.7 and Nav1.8 (values are summarized in Table 6).
Table 5.
Potencies for Inhibition of Nav1.9 and Nav1.9 F1592A/Y1599A.
Table 6.
Potencies for Inhibition of Nav1.7 and Nav1.8.
Fig 3.
Interaction of sodium channel inhibitors with Nav1.9 canonical local anesthetic binding site.
(A) Sequence alignment of D4 S6 segments for Nav1.1 to Nav1.9. The canonical local anesthetic binding site residues phenylalanine and tyrosine are labeled in magenta and were mutated to alanine (F1592A/Y1599A) to assess their contribution to Nav1.9 inhibition. (B) Comparison of the voltage dependence of activation (Nav1.9 WT: -51 ± 1 mV, n = 9; Nav1.9 F1592A/Y1599A: -56 ± 1 mV, n = 10; P>0.05) and inactivation following a 500 ms conditioning prepulse (WT: -50 ± 1 mV, n = 9; Nav1.9 F1592A/Y1599A: -56 ± 1 mV, n = 10; P<0.05). Comparison of concentration response relationships for inhibition of Nav1.9 WT (open symbols) and Nav1.9 F1592A/Y1599A (Closed symbols, LAM) by tetracaine (C), TC-N 1752 (D), and benzocaine (E). Fitted IC50s are summarized in Table 5.
Fig 4.
Characterization of K799 residue unique to Nav1.9 in Domain 2 S6.
(A) Upper panel; sequence alignment of D2 S6 for Nav1.1 to Nav1.9. Lysine residue K799 is labeled in blue whereas the consensus asparagine found at the equivalent position in other Nav channel subtypes is shown in green. Lower panel; sequence alignment of Nav1.9 D2 S6 with D4 S6 segments showing position of K799 residue relative to F1592 and Y1599 residues that form part of the canonical local anesthetic binding site. (B) Homology model of human Nav1.9 based on X-ray crystal structure of NavM [26] illustrating the position of lysine K799 in relation to F1592 and Y1599. (C) Comparison of the voltage dependence of activation (Nav1.9 WT: -51 ± 1 mV, n = 9; K799N: -48 ± 1 mV, n = 14; P>0.05) and inactivation induced by 500ms conditioning prepulse (Nav1.9 WT: -50 ± 1 mV, n = 9; K799N: -54 ± 1 mV, n = 14; P>0.05). (D) Comparison of concentration dependence of inhibition of Nav1.9 WT and Nav1.9 K799N channels by tetracaine, TC-N 1752 and benzocaine. Potency was determined using protocol described in Fig 2. (E) Comparison of the voltage dependence of activation and inactivation of Nav1.7 WT and Nav1.7 N945K mutant channels (activation; WT: -26 ± 0.4 mV, n = 10; N945K: -26 ± 0.4 mV, n = 9; P>0.05; and inactivation, WT: -65 ± 0.3 mV, n = 10; N945K: -64 ± 0.3 mV, n = 9; P>0.05) using same conditioning prepulse protocol as in (C) except utilizing a 20 ms test pulse to 0 mV. (F) Comparison of concentration dependence of inhibition of Nav1.7 WT and Nav1.7 N945K channels by tetracaine, TC-N 1752 and benzocaine.
Fig 5.
Expression and biophysical properties of rodent Nav1.9 stably expressed in HEK-293 cells.
Representative family of current traces recorded in HEK-293 cells stably expressing, (A) mouse Nav1.9 plus mouse β1/β2 or (B) rat Nav1.9 plus rat β1/β2. (C) Comparison of voltage dependence of activation for human, mouse and rat Nav1.9. (D) Comparison of steady-state inactivation following 100 ms conditioning prepulse for human, mouse and rat Nav1.9. Parameters for both activation and inactivation derived from fits of the Boltzmann equation are summarized in Table 2. (E) Comparison of concentration dependence of inhibition of human, mouse and rat Nav1.9 by tetracaine. (F) Comparison of concentration dependence of inhibition of human, mouse and rat Nav1.9 by TC-N 1752. Data points for (E) and (F) are means ± SEM for n = 4 to 13 observations. IC50s derived from fitting data are summarized in Table 7.
Table 7.
Potencies for Inhibition of Rat and Mouse Nav1.9.