Because cooling is known to alleviate symptoms in IEM [4
], a disorder caused by mutations in Nav1.7 [1
], we have investigated the effect of temperature on the gating behavior of WT channels and IEM mutant L858F Nav1.7 channels in HEK293 cells stably expressing these channels. Using whole-cell voltage-clamp, we show in this study that lowering the temperature of the recording solution causes a decrease in current density, an increase in ramp currents and a slowing in deactivation for both WT and mutant channels. The V1/2
of steady-state activation shows differential temperature sensitivity, and is depolarized at lower temperatures for L858F, but not for WT channels.
A temperature of 35°C was chosen as a reference because it is closest to physiological conditions. The normal temperature of human skin is ~34°C and it can be reduced quickly when exposed to cold water [21
]. While cell bodies of DRG neurons are located close to the spinal cord and are therefore at body temperature (~36°C), immunolabelling for Nav1.7 is present along unmyelinated fibers in situ [22
], and is predicted to accumulate distally within nerve terminals [11
] in the skin, where the channels are exposed to large variations in temperature.
All patients with IEM reported to date experience pain relief by cooling of the limbs. It is not known if all of the mutant Nav1.7 channels respond to cooling in a manner similar to the L858F channels described here. Lowering temperature would be expected to lead to a reduced rate of gating of Nav1.7 channels, as shown previously for neuronal sodium channels in myelinated axons and for muscle sodium channels [23
]. The decrease in current density that we observed with cooling can be explained at least in part by slowed channel gating. We propose that differential cold-induced modification of gating of mutant Nav1.7 can explain, at least in part, why cooling limbs helps to alleviate the pain.
The hyperpolarizing shift in the V1/2
of steady-state activation for L858F compared to WT, at all temperatures tested, is in agreement with our earlier findings [7
], and provides a possible explanation for increased excitability in DRG neurons expressing the IEM mutation. However, a reduction in temperature to 16°C causes a significant shift in a depolarizing direction of the V1/2
of activation of L858F channels, and this shift is not observed for WT channels. This differential effect causes the activation V1/2
for L858F to come closer to the V1/2
of WT channels at 16°C. Interestingly, the temperature effect on the slope factor of activation is the same for mutant and WT channels. Increasing the activation threshold of L858F channels is expected to result in a decrease in excitability in DRG neurons expressing the mutant channel, suggesting a possible contribution of this shift of V1/2
of activation of the mutant channel to alleviating the symptoms of IEM upon cooling the affected extremities. The V1/2
of steady-state fast inactivation, on the other hand, is influenced by temperature in the same way for WT and L858F channels; therefore, a contribution of the shifts in fast inactivation properties to pain alleviation induced by cooling seems unlikely.
Temperature changes have been shown to trigger clinical changes in patients harboring some mutations of the cardiac sodium channel (Nav1.5) and the skeletal muscle sodium channel (Nav1.4) [25
]. Changes in temperature, for example, evoke symptoms in two inherited diseases of sodium channels, with fever triggering cardiac arrhythmia in Brugada syndrome [27
] and cold exacerbating muscle weakness or myotonia in paramyotonia congenita [29
]. The V1/2
of activation of WT Nav1.5 does not show temperature sensitivity [32
], similar to our findings for WT. The V1/2
of activation of WT Nav1.4, however, has been reported to shift in a depolarizing direction upon cooling [33
]. One mutation of Nav1.4 causing paramyotonia congenita (I693T [34
]) is located in the S4–S5 linker of domain II, only 10 amino acids N-terminal to the amino acid substitution in L858F in Nav1.7 [7
], and is located at corresponding position to the IEM mutation Nav1.7/I848T [15
]. Patients with the paramyotonia I693T Nav1.4 mutation, and those with the erythromelalgia mutation Nav1.7, both show temperature sensitivity; but while cooling of the IEM patients with Nav1.7/I848T mutations relieves pain, it precipitates symptoms in paramyotonia patients carrying the Nav1.4/I693T mutation. It is interesting in this regard that Plassart-Schiess et al. [33
] showed that a reduction in temperature produced a similar effect on WT Nav1.4 and Nav1.4/I693T. Taken together, these data suggest that temperature effects on the behavior of excitable cells may depend on the differential sensitivity of voltage-gated sodium channels and other ionic conductances, which are expressed in these cells.
Nav1.7 responds to slow depolarizations with ramp currents at potentials that are hyperpolarized relative to the threshold of action potential firing, and thus appears to amplify stimuli that, in themselves, do not reach the threshold for the generation of action potentials [15
]. L858F has been reported to significantly increase ramp current [7
]. We observed this larger ramp current of L858F compared to WT channels at every temperature tested. With cooling, the size of the ramp current increases for both mutant and WT channels. Because the ramp current appears to boost small subthreshold depolarizations [20
], we were surprised to see that the increase in ramp current with cooling appeared in mutant as well as in WT channels. As we have noted previously [1
] multiple factors, including shift in the voltage-dependence of activation can contribute to hyperexcitability of DRG neurons that express mutant Nav1.7 channels. Thus, we speculate that the depolarizing shift in voltage-dependence of activation of L858F Nav1.7 channels at 16°C, which is predicted to decrease DRG neuron excitability, outweighs the effect of the increased ramp currents.
It should be noted that we have recently shown that the presence of Nav1.8 is critical for rendering mutant Nav1.7 channels expressing DRG neurons hyperexcitable [18
]. Nav1.7 appears to be responsible for the initiation of the action potential, whereas the current which underlies the upstroke of action potential is contributed mainly by Nav1.8 [35
]. The effects of cooling on Nav1.8 are not well understood at this time, but it is possible that altered biophysical properties of Nav1.8, along with altered properties of Nav1.7, contribute to alleviation of pain at decreased temperatures in IEM.