PF-06526290 can both enhance and inhibit conduction through voltage-gated sodium channels

Abstract

Background and purpose: Pharmacological agents that either inhibit or enhance flux of ions through voltage-gated sodium (Na_v ) channels may provide opportunities for treatment of human health disorders. During studies to characterize agents that modulate Na_v 1.3 function, we identified a compound that appears to exhibit both enhancement and inhibition of sodium ion conduction that appeared to be dependent on the gating state that the channel was in. The objective of the current study was to determine if these different modulatory effects are mediated by the same or distinct interactions with the channel.

Experimental approach: Electrophysiology and site-directed mutation were used to investigate the effects of PF-06526290 on Na_v channel function.

Key results: PF-06526290 greatly slows inactivation of Na_v channels in a subtype-independent manner. However, upon prolonged depolarization to induce inactivation, PF-06526290 becomes a Na_v subtype-selective inhibitor. Mutation of the domain 4 voltage sensor modulates inhibition of Na_v 1.3 or Na_v 1.7 channels by PF-06526290 but has no effect on PF-06526290 mediated slowing of inactivation.

Conclusions and implications: These findings suggest that distinct interactions may underlie the two modes of Na_v channel modulation by PF-06526290 and that a single compound can affect sodium channel function in several ways.

Figure 1

PF‐06526290 slows Na_v channel inactivation. (A) Structure of PF‐06526290. (B) Voltage protocol employed to evaluate PF‐06526290 effect on sodium channel function. (C) Current traces for the first 10 ms (left) or 500 ms (right) of the 5 s voltage step to 0 mV, showing the effect of PF‐06526290 on human Na_v1.3 and Na_v1.7 channel activity. (D) Concentration dependence of human Na_v1.3 and Na_v1.7 slowed inactivation following PF‐06526290 treatment. Magnitude of PF‐06526290‐induced slowing of inactivation was calculated by normalizing sodium current amplitude 5 ms after peak‐to‐peak amplitude; EC₅₀ 0.27 ± 0.08 μM (n = 5) for Na_v1.7 and 1.1 ± 0.1 μM (n = 6) for Na_v1.3 channels. Data values for Na_v1.7 channels derived from 21 cells from nine separate cell preparations. Data values for Na_v1.3 channels derived from 20 cells from eight separate cell preparations.

Figure 2

(A) Time course of 10 μM PF‐06526290 slowing of inactivation of hNa_v1.7 channels and washout. Time course of Na_v1.7 5 ms/peak current ratio was normalized to the maximal response in each experimental run just prior to compound washout. Data shown are mean ± SEM for five to six separate experiments. (B) Sodium channel auxiliary subunits β1 and β2 have no effects on PF‐06526290 (PF‐290) induced slowing of inactivation or inhibition. Representative current traces of Na_v1.7 + β1/β2 subunits with and without 10 μM PF‐06526290 induced by a two‐pulse test protocol as shown in Figure 1. (C) Co‐expression of β1/β2 subunits with Na_v1.7 had no effect on PF‐06526290 induced slowing of inactivation (I_5ms/I_peak) compared to Na_v1.7 α subunit only expressing cells. Individual data for Na_v1.7 β1/β2 from seven separate cells from two different cell preparations. Individual data for Na_v1.7 from nine separate cells from three different cell preparations. *P < 0.05, significantly different as indicated; N.S., not significant; ANOVA.

Figure 3

(A) Effect of 10 μM PF‐06526290 on current amplitude and rate of inactivation of human Na_v1.1, Na_v1.2, Na_v1.4, Na_v1.5, Na_v1.6 and Na_v1.8 sodium currents elicited by Pulse 1 of the protocol illustrated in Figure 1B. (B) For each sodium channel subtype, I_5ms/I_Peak ratio before (left‐hand data set) and after (right‐hand data set) application of 10 μM PF‐06526290 is shown. Each pair of data points for individual channel subtypes represent a separate cell recording. Data points are from 5 to 14 separate cells from two to three different cell preparations for each channel type except Na_v1.5 which is from four separate cells from one cell preparation. *P < 0.05, significantly different as indicated; t‐test.

Figure 4

Effect of PF‐06526290 on voltage dependence of activation and inactivation of Na_v1.3 and Na_v1.7 channels. (A, B) Normalized peak current amplitude versus stimulating voltage, for hNa_v1.3 and hNa_v1.7 channels in the absence and presence of 10 μM PF‐06526290. Control currents were normalized to maximum current amplitude in the absence of PF‐06526290, while compound effect on current amplitude for each cell was normalized to the maximal control current for that cell. Data shown are mean ± SEM for four to five separate cell recordings. (C, D) Voltage dependence of activation and inactivation of Na_v1.3 and Na_v1.7 channels in the absence and presence of 10 μM PF‐06526290. Normalized conductance in the presence of PF‐06526290 was measured 5 ms after peak current. [hNa_v1.3: Activation V_1/2: −12 ± 1, k: 7 ± 1.0 (n = 5); inactivation V_1/2: −52 ± 1, k: 6 ± 1 (n = 5); With 10 μM PF‐06526290: Activation V_1/2: −19 ± 1, k: 6 ± 1 (n = 5); Inactivation V_1/2: −37 ± 2, k: 13 ± 2 (n = 5). hNa_v1.7: Activation V_1/2: −20 ± 1, k: 6 ± 1 (n = 4); Inactivation V_1/2: −70 ± 0.6, k: 6 ± 0.5 (n = 5); With 10 μM PF‐06526290: Activation V_1/2: −32 ± 1, k: 6 ± 1 (n = 3); Inactivation V_1/2: −32 ± 1, k: 6 ± 1) (n = 6)]. Data generated from two separate cell preparations. (E) Voltage dependence of inactivation of Na_v1.7 in the presence of 0.1, 1 and 10 μM PF‐06526290 (n = 5–6 cells from two separate cell preparations). Each data set was fitted to a double Boltzmann Equation where each of the V_1/2 and slope of inactivation parameters were fixed to either the control or 10 μM PF‐06526290 values shown in (D). However, the relative proportion of current with either the −70 or −32 mV V_1/2 was adjusted to give the best fit for each concentration of PF‐06526290 tested.

Figure 5

PF‐06526290 produces subtype‐selective inhibition of Na_v channels following prolonged depolarization. (A) Comparison of PF‐06526290 effects on Na_v1.3 and Na_v1.7 current traces elicited by protocol shown in (B). Whereas slowing of inactivation by 10 μM PF‐06526290 is observed for both channel subtypes with Pulse 1 only inhibition of Na_v1.3 current was observed with Pulse 2. Furthermore, neither Na_v1.7 nor Na_v1.3 current traces elicited by Pulse 2 exhibited the slowing of inactivation observed with Pulse 1 (for Na_v1.3, the blue trace reflects the uninhibited component scaled to control). (C) Concentration dependence of human Na_v1.3 and Na_v1.7 inhibition by PF‐06526290 [IC₅₀ 5 ± 2 μM (n = 6 cells from two cell preparations) for Na_v1.3 channels and > 30 μM (n = 6 cells from two cell preparations) for Na_v1.7 channels]. (D) Inhibition effect of PF‐06526290 on different Na_v channel subtypes tested. For each sodium channel subtype, data before (left‐hand data set) and after (right‐hand data set) application of 10 μM PF‐06526290 are shown. *P < 0.05; significantly different as indicated; Student's t‐test.

Figure 6

Properties of PF‐06526290 induced slowing of Na_v channel inactivation. (A) Current traces of Na_v1.7 channels in the absence and presence of 0.1, 1 and 10 μM PF‐06526290. Sodium currents were elicited by voltage steps to 0 mV for 500 ms from a holding potential of −120 mV (current amplitudes are normalized to peak for each trace). (B) Same sodium current traces from (A) normalized to peak and plotted on a log scale. (C) Time constant of inactivation (τ) in the absence and presence of 0.1, 1 and 10 μM PF‐06526290 (n = 4–7 cells from four cell preparations). Current decay was fit with a single exponential. There was no significant difference between the calculated time constants for slow phase of inactivation at different concentrations of PF‐06526290. *P < 0.05, significantly different from control; One way ANOVA with Tukey's post hoc test (D) Na_v1.7 current trace evoked by a single pulse depolarization to 0 mV for 20 ms from a holding potential of −120 mV the in presence of 1 μM PF‐06526290. (E) Current traces elicited by the voltage protocol shown in the presence of 1 μM PF‐06526290. A 500 ms depolarizing voltage step to 0 mV was applied to functionally displace PF‐06526290. A rest period at −120 mV of variable duration was applied prior to a 20 ms test pulse (indicated by #) to assess fraction of current exhibiting slowed inactivation. (F) Concentration dependence of time course for recovery of slowed inactivation after 500 ms voltage step to 0 mV in the presence of PF‐06526290. The I_5ms/I_peak current amplitude ratios determined during the test pulse (#) are plotted against time at −120 mV after 500 ms voltage step to 0 mV. τ_Recovery was determined from a least squares fit of a double exponential with the slow phase contributing 92–94% of the recovery for each concentration and τ_slow being 2311 ± 18 ms (n = 6), 994 ± 121 ms (n = 6) 540 ± 72 ms (n = 6) and 363 ± 63 ms (n = 5) for 0.3, 1, 3 and 10 μM PF‐06526290 respectively. For comparison, the time course for recovery of Na_v1.7 inactivation following a 500 ms voltage step to 0 mV in the absence of PF‐06526290 is also shown (Control). Data were fit with a double exponential with τ_fast = 11 ± 2, τ_slow = 270 ± 128 ms and fast/slow ratio of 0.74. Results shown in (D–F) were obtained using Molecular Devices PatchXpress automated patch clamp platform.

Figure 7

(A) Introduction of M123 (S1510Y/R1511W/E1559D) residues into Na_v1.7 increases sensitivity to inhibition by PF‐06526290 whereas introduction of M123 (Y1537S/W1538R/D1586E) residues into Na_v1.3 reduces its sensitivity to inhibition by PF‐06526209 [IC₅₀: 5 ± 2 μM (n = 6 cells) for Na_v1.3, 30 ± 5 μM (n = 3 cells) for Na_v1.3 M123, >100 μM (n = 6 cells) for Na_v1.7 and 35 ± 4 μM (n = 3 cells) for Na_v1.7 M123] (from at least two separate cell preparations). (B) Mutation of M123 residues has no effect on PF‐06526290‐induced slowing of inactivation in either Na_v1.3 or Na_v1.7 [EC₅₀: 1.1 ± 0.1 μM (n = 5 cells) for Na_v1.3, 1.1 ± 0.4 μM (n = 4 cells) for Na_v1.3 M123, 0.27 ± 0.08 μM (n = 6 cells) for Na_v1.7, and 0.39 ± 0.13 μM (n = 3 cells) for Na_v1.7 M123] (from at least two separate cell preparations). (C) Na_v1.7 current traces recorded in the presence of 300 nM scorpion toxin Lqh III, 1 μM PF‐06526290 (PF‐290) or mixture of 300 nM Lqh III + 1 μM PF‐06526290. Currents were elicited by a 5 s voltage step from −120 to 0 mV (traces show the first 500 ms). (D) Plot of inactivation time constants (τ_inact) in the absence and presence of 300 nM Lqh III (n = 6), 1 μM PF‐06526290 (PF‐290) (n = 5) or a mixture of 300 nM Lqh III + 1 μM PF‐06526290 (n = 9). τ_inact was determined from a fit of a single exponential equation. Data derived from three separate cell preparations. *P < 0.05, significantly different from control; One way ANOVA with Tukey's post hoc test. (E) Current traces comparing effect of 10 μM PF‐06526290 on inactivation of Na_v1.7 versus the local anaesthetic binding site mutant Na_v1.7 F1737A/Y1744A. (F) Plot of I_5ms/I_peak current amplitude ratio for both Na_v1.7 and Na_v1.7 F1737A/Y1744A in the presence and absence of 10 μM PF‐06526290 Data shown are from six to nine separate experiments. *P < 0.05, significantly different as indicated; N.S., not significant; One way ANOVA with Tukey's post hoc test.

Figure 8

PF‐06526290 slows inactivation of endogenous Na_v currents and increases neuronal excitability in mouse sensory neurons. (A) Sodium current traces showing the slowing of inactivation by 10 μM PF‐06526290 in the absence or presence of 200 nM TTX to isolate the TTX‐resistant component. Sodium currents were elicited by a single pulse test to 0 mV for 20 ms from a holding potential of −120 mV. (B) Plot of the I_5ms/I_peak current amplitude ratio for TTX‐sensitive and ‐resistant currents in the presence of 10 μM PF‐06526290 (n = 8–10 neurons recorded over 3 days from two separate cell isolations). *P < 0.05, significantly different as indicated; N.S., not significant; One way ANOVA with Tukey's post hoc test. (C) Effect of 0.3 μM PF‐06526290 on stimulus intensity (current injection in pA) required to initiate action potential. (D) Plot of current injection threshold for initiation of action potential in the presence and absence of 0.3 μM PF‐06526290 (n = 6 neurons recorded over 3 days from two separate cell isolations). *P < 0.05, significantly different as indicated; N.S., not significant; Student's t‐test. (E) Time‐dependent change in number of action potentials elicited by a 500 ms 150 pA supramaximal stimulus at 0.1 Hz in the absence or following administration of 0.3 μM PF‐06526290. Plot time course of action potential frequency (F) or duration at 50% repolarization (G), in the absence or following administration 0.3 μM PF‐06526290 using stimulus protocol used in (E) (n = 6 neurons recorded over 2 days from two separate cell isolations).