A Novel Gain-of-Function Nav1.9 Mutation in a Child With Episodic Pain

Figures

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  FIGURE 1. Schematic illustration of human voltage-gated sodium channels and location of the N816K mutation. A functional Nav channel consists of four domains (DI-DIV) and each domain consists of six transmembrane segments (S1–S6). Intracellular loop L2 connects DII to DIII. Sequence alignment of DII/S6 and beginning of L2 from all human Navs shows the location of the N816K mutation. N816K mutation (red) is separated by one residue from the gating phenylalanine residue (blue), which contributes to the hydrophobic ring at the cytoplasmic aperture of Nav channels, stabilizing the closed state of the channel (Lampert et al., 2008). Previous studies reported two gain-of-function mutations, A808G (Zhang et al., 2013) and L811P (Leipold et al., 2013), in hNav1.9 channels residing in DII/S6, which are marked with asterisks.

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  FIGURE 2. N816K mutation hyperpolarizes voltage-dependence of activation of hNav1.9 channels in small DRG neurons. Representative traces showing characteristic human Nav1.9 currents from small DRG neurons expressing (A) wild-type (WT) or (B) N816K mutant Nav1.9 channels in adult Nav1.9-null mice. (C) Scatter plot of current densities, determined by normalization of peak current to cell capacitance, in small DRG neurons expressing WT and N816K mutant hNav1.9 channels. Solid lines indicate mean current densities. ∗p < 0.05. (D) Current-voltage relationships (I–V curves) for WT and N816K mutant hNav1.9 channels. Normalized peak inward current was plotted against voltage commands. N816K mutation hyperpolarizes the I–V curve of Nav1.9 channels. Boltzmann fits for activation (E) and steady-state fast-inactivation (F) show that N816K hyperpolarizes voltage-dependence of activation of Nav1.9 channels, but does not significantly change voltage-dependence of steady-state fast inactivation.

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  FIGURE 3. N816K mutation depolarizes resting membrane and reduces threshold for action potentials in small DRG neurons. (A) A representative 30 s trace from a cell expressing N816K mutant channels demonstrates spontaneous firing of action potentials (left panel). However, N816K mutant channels do not change the proportion of spontaneously-firing cells, compared to WT hNav1.9 channels (right panel). (B) Scatter plot of resting membrane potential (RMP) in small DRG neurons expressing WT or N816K mutant hNav1.9 channels. Solid lines indicate mean RMP. ∗∗∗p < 0.001. (C) Scatter plot of current threshold, the minimal current injection required to elicit an action potential in small DRG neurons expressing WT or N816K mutant hNav1.9 channels. Solid lines indicate mean RMP. ∗∗∗p < 0.001. (D) Representative action potential traces recorded from a small DRG neuron expressing WT hNav1.9 channels show that the cell produces subthreshold depolarizations in response to current stimuli from 150 to 195 pA, and does not generate an all-or-none action potential until the injected current reaches 200 pA, which is defined as its current threshold. (E) Representative action potential traces recorded from a…

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  FIGURE 4. N816K mutation enables hyperexcitability in small DRG neurons. (A) Scatter plot of maximal numbers of action potentials evoked by stimuli ranging from 25 to 500 pA in cells expressing WT and N816K mutant hNav1.9 channels. Cells are divided into two groups, those that only fire a single action potential during 500 ms course and those that fire multiple action potentials. N816K mutation increases the proportion of cells that fire multiple action potentials, compared to WT hNav1.9 channels. (B) Average action potential frequency during 500 ms current injections ranging from 25 to 500 pA in 25 pA increments. ∗p < 0.05.

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  FIGURE 5. A small DRG neuron expressing N816K mutant hNav1.9 channels produces more action potentials than WT in response to the same stimuli. Representative action potential traces from a small DRG neuron expressing (A) WT or (B) N816K mutant hNav1.9 channels in response to 200, 350, and 500 pA current stimuli, respectively.