PMC5012686_01
ADS during 2 Hz stimulation protocol of CMi-nociceptors in the M650K patient (A) and EM controls without Nav mutations (B) and age-matched healthy controls (C).. The lower panel shows the first 15 pulses of the upper panel (subset indicated with gray shadow in the upper panel). Note the prominent abnormal progressive increase in ADS in the last part of the stimulation in two of the CMi-nociceptors from the M650K patient (red), none in EM patients without mutation and only one less pronounced in age-matched healthy controls showed this behavior.
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However, in contrast to what was displayed during the low frequency protocol, the CMi-nociceptors of the patient carrying the M650K mutation displayed more ADS during 2 Hz stimulation compared to the EM-controls and healthy subjects, even during the first pulses of the protocol (Table 4 and Fig 2A-2C, lower panel). Two CMi-fibers of the M650K patient showed an abnormal increase in ADS at the end of the high-frequency protocol (Fig 2A).
The M650K mutation decreases the firing rate of DRG neurons.. A: Schematic overview of a eukaryotic Nav showing the four domains DI to DIV, each consisting of the transmembrane segments S1 to S6. The rare genetic variant M650K in Nav1.8 (red dot,) is located at the adjacent intracellular side of DII/S1. B: Number of APs evoked by 500 ms square step current injections to WT (black markers) or M650K (red markers) transfected cells (n = 18 and 19). * < 0.05 in a 2-sided t-test C: Example of an AP recorded from a WT (black trace) and a M650K (red trace) transfected DRG neuron evoked by a 5 ms depolarizing pulse. Injected current was twice threshold current. D: The AP half width was significantly broader in M650K than in WT transfected neurons (n = 13 and 19). *< 0.05 in a 2-sided t-test.
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The mutation M650K which was found in our patient is located at the intracellular side of DIIS1 in the linker between DI and DII of Nav1.8 (Fig 3A). Surprisingly, neurons transfected with the M650K mutation produced significantly fewer APs compared to WT when stimulated with a 500 ms square current pulse (Fig 3B). Single step evoked APs revealed that transfection of the M650K mutation broadened the AP half width at 0 mV from 4.9 +- 0.4 ms to 7.0 +- 0.9 ms (Fig 3C and 3D, p<0.05 in a two sided t-test).
Voltage-dependence of activation and deactivation of Nav1.8 is unaltered by the M650K mutation.. A: Pulse protocol and representative voltage-clamp recordings of hNav1.8 WT and hNav1.8 M650K expressed heterologously in ND7/23 cells. Traces were digitally low pass filtered for display.B: Conductance-voltage relations for WT (black circles, n = 10) and the Nav1.8 M650K mutation (red circles, n = 12) are shown. Solid lines represent Boltzmann-fits to the mean data points. C: Pulse protocol for deactivation measurements and representative recordings of hNav1.8 WT and hNav1.8 M650K mutant expressed heterologously in ND7/23 cells. D: Deactivation time constants as derived from single exponential fits of current decay during repolarization for WT (black circles, n = 13) and M650K (red circles, n = 15) at various voltages.
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By adding 1 muM TTX to the bath solution we blocked all endogenous Nav currents and thus recorded Nav1.8 in isolation using voltage-clamp (Fig 4). Nav1.8 activated and inactivated within 20 ms (Fig 4A) and introduction of the M650K mutation did not alter voltage-dependence of activation (Fig 4B). WT and M650K, respectively. Broader APs like those recorded in M650K transfected DRG neurons may be due to slowed deactivation kinetics. We assessed deactivation using a brief depolarization of 1 ms to +20 mV in order to fully activate all expressed channels, followed by hyperpolarizing pulses to potentials between -120 mV and 0 mV (Fig 4C). Time constants derived from a single exponential fit to the deactivating currents were not significantly different between WT and M650K mutation at all potentials tested (Fig 4D). Inactivation decay kinetics, as determined by a single exponential fit to current decay using depolarizing pulses (as shown in Fig 4A) did not show any differences between WT and the M650K mutation (Fig 5C).
The M650K mutation shifts steady-state fast inactivation to more hyperpolarized potentials.. A: Voltage dependence of steady-state fast inactivation as determined with the indicated pulse protocol. Black circles indicate WT (n = 7-8) and red circles the M650K mutation (n = 7-8). B: Distribution of the Vhalf determined by Boltzmann fits to each cell. Mean is indicated as an open square, the median is depicted as a line inside the box, the boxes indicate 50% confidence interval and whiskers indicate 95% confidence interval. C: Time constants of single exponential fits of currents evoked with the pulse protocol shown in Fig 4A did not differ between WT and M650K mutation at any investigated voltage.
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In contrast, the voltage-dependence of steady-state fast inactivation was significantly shifted to more hyperpolarized potentials (Fig 5A). Mean Vhalf revealed by Boltzmann-fits for WT and M650K mutant were -66.0 mV +- 6.5 mV and -73.8 mV +- 2.2 mV (slope: -9.03 mV and -9.66 mV), respectively (Fig 5B). Inactivation decay kinetics, as determined by a single exponential fit to current decay using depolarizing pulses (as shown in Fig 4A) did not show any differences between WT and the M650K mutation (Fig 5C).