The electrophysiological properties of wild-type (WT) or mutant NaV
channels were investigated using whole-cell voltage-clamp recording in HEK293 cells stably expressing WT, P1308L (IEM), or V1298F (PEPD) channels. Figure shows representative inward Na+
currents recorded from cells stably expressing WT, P1308L, or V1298F NaV
channels. Pilot experiments showed that cells expressing mutant channels produced smaller currents than cells expressing WT channels. To determine whether the reduced P1308L current density is the result of intrinsic effects of the mutations or the effect of the site of integration of the channel within the HEK 293 genome, we measured the sodium current density in transiently transfected HEK 293 cells. V1298F has previously been reported [25
] to produce smaller currents than WT channels in transiently transfected HEK 293 cells, and we did not follow up on it in this study. The current density of P1308L in transiently-transfected HEK 293 cells is significantly smaller than that of WT channels (WT: 285 ± 46 pA/pF, n = 9; P1308L: 90 ± 14, n = 11, p
= 0.003, two-tailed student's t test). To examine whether the smaller current densities of mutant channels P1308L and V1298F were due to lower protein expression, we used Western blot to assay channel protein levels in transiently transfected HEK 293 cells. When compared with WT channels (set as 100%), the protein levels of mutant channels were 85 ± 12% (n = 3, p
= 0.526) for P1308L and 123 ± 15% (n = 2, p
= 0.352) for V1298F channels (Figure ), suggesting that the smaller currents from mutant channels are not caused by reduced channel synthesis in HEK 293 cells.
Figure 2 P1308L and V1298F exhibit different effects on voltage-dependence of activation and fast inactivation. A, Representative families of traces of Na+ currents (INa) from voltage-clamped HEK293 cells stably expressing wild-type (WT), P1308L, or V1298F Na (more ...)
To minimize the difference of voltage error between WT and mutant P1308L currents, HEK 293 cells producing currents larger than 10 nA (i.e. representing ≥ 4X the mean peak currents of P1308L channels) were excluded from the analysis; none of the cells expressing P1308L or V1298F channels produced sodium currents larger than 10 nA. Despite the exclusion of cells producing large WT currents, the current densities of mutant channels were still significantly smaller than that of WT channels (WT: 351 ± 25 pA/pF, n = 29; P1308L: 146 ± 15 pA/pF, n = 25, p < 0.001 vs WT; V1298F: 183 ± 16 pA/pF, n = 26, p < 0.001 vs WT; non-parametric Kruskal-Wallis statistical test).
Like all IEM mutant channels characterized thus far, P1308L mutation caused a hyperpolarizing shift (-9.6 mV) of activation (WT: V1/2,act = -21.8 ± 0.7 mV, k = 6.95 ± 0.11, n = 29; P1308L: V1/2,act = -31.4 ± 0.5 mV, k = 7.33 ± 0.09, n = 25; p < 0.001 for V1/2,act and p = 0.012 for k) whereas V1298F, the PEPD mutation, had no effect on activation (V1/2,act = -22.5 ± 0.7 mV, p = 0.680, and k = 6.83 ± 0.07, p = 0.754, n = 26) (Figure and , Table ). P1308L mutation did not affect the midpoint (V1/2,fast) of steady-state fast-inactivation, but altered the slope of fast-inactivation curve (WT: V1/2,fast = -80.6 ± 1.1 mV, k = 6.32 ± 0.14, n = 13; P1308L: V1/2,fast = -78.9 ± 0.7 mV, p = 0.422, k = 5.65 ± 0.11, p = 0.002) (Figure , Table ). Like other PEPD mutations characterized to date, V1298F channels showed a depolarizing shift (+16.1 mV) of steady-state fast-inactivation and a steeper inactivation curve (V1/2,fast = -64.5 ± 0.9 mV, p < 0.001, k = 5.45 ± 0.15, p < 0.001, n = 13) (Figure , Table ).
Parameters of voltage-dependent activation and steady-state fast-inactivation of WT, P1308L, and V1298F NaV 1.7R channels.
Activation kinetics (measured as time-to-peak) of P1308L channels were faster at -20 and -15 mV, whereas V1298F mutant channels were slower (between -20 to +40 mV) compared to WT channels (Figure ). The kinetics of open-state fast-inactivation were analyzed by mono-exponential fit of the decaying phase of Na+
currents in Figure . V1298F mutant channels exhibited slower inactivation kinetics, compared to WT, from -25 to +40 mV, whereas P1308L channels showed faster kinetics at -30 and -25 mV (Figure ). The faster inactivation kinetics of P1308L may be due to enhanced activation of P1308L channels, since open-state fast-inactivation is coupled to the activation state of channels [27
Deactivation kinetics reflect the transition of channels from the open state to the closed state. As with most IEM mutations, P1308L mutant channels showed slow deactivation of Na+ currents at all potentials tested, indicating that the mutant channel resides longer in the open state, whereas V1298F mutation had no effect on deactivation kinetics (Figure ).
Figure 3 The P1308L and V1298F mutations have different effects on channel deactivation and slow-inactivation. A, To measure deactivation kinetics, cells were held at -100 mV and tail currents were generated by a brief 0.5-ms depolarization to -20 mV followed (more ...)
Steady-state slow-inactivation develops over a long time frame (from seconds to minutes) upon sustained stimulation. The slow-inactivation of sodium channels was evaluated using 30-s prepulses at potentials ranging from -130 to +10 mV. P1308L did not significantly affect the voltage-dependence of slow-inactivation of mutant channels (WT: V1/2,slow = -63.8 ± 1.7 mV, n = 14; P1308L: V1/2,slow = -68.4 ± 1.2 mV, n = 10; p = 0.072, Table , Figure ), while V1298F depolarized the slow-inactivation curve of mutant channels by +6 mV (V1298F: V1/2,slow = -57.8 ± 1.1 mV, n = 12; p = 0.011, Table , Figure ). Both mutant channels decreased the slope factor of the inactivation curve, and increased the fraction of channels resistant to slow inactivation (Rresist, expressed as % of maximal current and calculated as offset (A) × 100%. For WT: k = 12.6 ± 0.3, Rresist = 14.0 ± 1.2%, n = 14; P1308L: k = 10.5 ± 0.3, p < 0.001, Rresist = 19.1 ± 1.4%, n = 10, p = 0.03; V1298F: k = 9.2 ± 0.3, p < 0.001, Rresist = 25.6 ± 1.5%, n = 12, p < 0.001) (Table , Figure ).
Parameters of steady-state slow-inactivation of WT, P1308L, and V1298F NaV1.7R channels.
Repriming kinetics reflect the recovery rate of channels from fast-inactivation state, and in the case of NaV
1.7, repriming kinetics may regulate how fast a neuron can repetitively fire [28
]. The P1308L mutation had no significant effect on recovery fractions or on repriming kinetics at tested potentials (Figure , and ). In contrast, V1298F mutant channels showed faster repriming kinetics at all tested potentials (Figure ) and higher recovery fraction at recovery potentials from -90 to -60 mV (Figure and ). The larger recovered fraction of V1298F channels is related to the depolarizing shift of fast-inactivation, which increases the proportion of channels available for activation at these recovery potentials. Faster repriming kinetics and larger recovery fraction of V1298F channels are expected to endow neurons housing this mutation with the ability to fire at higher frequency compared to wild type channels.
Figure 4 The P1308L and V1298F mutations have different effects on repriming. Cells were held at -100 mV, and fast-inactivation was initiated by a 20-ms depolarization to 0 mV, followed by a recovery period (2-300 ms) at a recovery potential. The available channels (more ...)
1.7 channels activate in response to small, slow depolarization, which allows them to amplify weak stimuli, e.g. generator potentials, bringing the membrane potential closer to the threshold for initiation of action potentials [11
]. Therefore, we examined the effects of mutations on the channel response to slow ramp depolarization (600 ms ramp depolarization from -100 mV to +20 mV; 0.2 mV/ms). Figure shows representative ramp currents recorded from cells expressing WT, P1308L and V1298F channels. The ramp currents, measured as percentage of peak current, generated by P1308L channels were about 4X larger than those of WT channels (WT: Iramp
= 0.26 ± 0.03%, n = 12; P1308L: Iramp
= 1.09 ± 0.11%, n = 15, p < 0.001) (Figure ). Consistent with the hyperpolarized shift of voltage-dependence of activation, P1308L mutation also shifted the peak of the ramp currents to more negative potentials (WT: Vramp
= -44.8 ± 1.4 mV, n = 12; P1308L: Vramp
= -51.1 ± 0.9 mV, n = 15, p < 0.001) (Figure ). Compared to WT channels, V1298F channels also produced 2X larger ramp currents, but had no effect on the voltage for peak ramp currents (V1298F: Iramp
= 0.58 ± 0.06%, n = 16, p
= 0.015; Vramp
= -42.5 ± 0.8 mV, n = 16, p
= 0.280) (Figure ).
Figure 5 The P1308L and V1298F mutations enhance the response to slow ramp depolarization. HEK293 cells were held at -100 mV and a depolarizing voltage ramp from -100 mV to +20 mV was applied at a rate of 0.2 mV/ms. A, Representative ramp currents from WT (black), (more ...)
To examine the effects of the P1308L and V1298F mutations on DRG neuron excitability, current-clamp recordings were performed on neonatal rat DRG neurons transfected with WT, P1308L, or V1298F constructs combined with GFP. The input resistance (Rinput), resting membrane potential (RMP), current threshold of action potential, and firing frequency in small DRG neurons were examined. Expression of mutant channels did not change Rinput and RMP of DRG neurons (Table ). However, expression of P1308L channels decreased the current threshold of action potential in DRG neurons (WT: 188 ± 14 pA, n = 38; P1308L: 122 ± 10 pA, n = 50, p < 0.001, Table , Figure ), whereas the action potential threshold of DRG neurons expressing V1298F channels was not significantly different from that of neurons expressing WT channels (V1298F: 154 ± 18, n = 27, p = 0.215, Table , Figure ).
Current clamp properties of DRG neurons transfected with WT, P1308L, or V1298F NaV1.7R construct.
Figure 6 Excitability is increased in DRG neurons transfected with the P1308L or V1298F mutant channels. A, Responses of DRG neurons expressing WT, P1308L, or V1298F channels to a series of current stimuli with 5-pA increment. DRG neurons expressing P1308L channels (more ...)
Previous studies have shown that DRG neurons are able to fire repetitively in response to sustained depolarizing stimuli [29
]. In this study, 11 out of 38 (29%) small DRG neurons expressing WT NaV
channels produced 3 or more action potentials, whereas a larger proportion of neurons expressing mutant channels were able to fire 3 or more spikes (64% for P1308L, and 78% for V1298F). Figure shows the mean firing frequency of DRG neurons in response to a series of 1-s current injections ranging from 25 to 500 pA in 25 pA increments, and Figure shows the responses elicited after injecting currents approximately 1.5X and 2X the threshold from the same neurons. Both P1308L and V1298F increased the firing frequency in transfected DRG neurons.