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. 2021 May 25:15:640709.
doi: 10.3389/fncel.2021.640709. eCollection 2021.

Overexpressed Na V 1.7 Channels Confer Hyperexcitability to in vitro Trigeminal Sensory Neurons of Ca V 2.1 Mutant Hemiplegic Migraine Mice

Riffat Mehboob  1   2 , Anna Marchenkova  1 , Arn M J M van den Maagdenberg  3   4 , Andrea Nistri  1
Affiliations

Overexpressed Na V 1.7 Channels Confer Hyperexcitability to in vitro Trigeminal Sensory Neurons of Ca V 2.1 Mutant Hemiplegic Migraine Mice

Riffat Mehboob et al. Front Cell Neurosci. .

Abstract

Trigeminal sensory neurons of transgenic knock-in (KI) mice expressing the R192Q missense mutation in the α1A subunit of neuronal voltage-gated Ca V 2.1 Ca2+ channels, which leads to familial hemiplegic migraine type 1 (FHM1) in patients, exhibit a hyperexcitability phenotype. Here, we show that the expression of Na V 1.7 channels, linked to pain states, is upregulated in KI primary cultures of trigeminal ganglia (TG), as shown by increased expression of its α1 subunit. In the majority of TG neurons, Na V 1.7 channels are co-expressed with ATP-gated P2X3 receptors (P2X3R), which are important nociceptive sensors. Reversing the trigeminal phenotype with selective Ca V 2.1 channel inhibitor ω-agatoxin IVA inhibited Na V 1.7 overexpression. Functionally, KI neurons revealed a TTX-sensitive inward current of larger amplitude that was partially inhibited by selective Na V 1.7 blocker Tp1a. Under current-clamp condition, Tp1a raised the spike threshold of both wild-type (WT) and KI neurons with decreased firing rate in KI cells. Na V 1.7 activator OD1 accelerated firing in WT and KI neurons, a phenomenon blocked by Tp1a. Enhanced expression and function of Na V 1.7 channels in KI TG neurons resulted in higher excitability and facilitated nociceptive signaling. Co-expression of Na V 1.7 channels and P2X3Rs in TGs may explain how hypersensitivity to local stimuli can be relevant to migraine.

Keywords: calcium channel; migraine; nociception; purinergic receptors; sodium channel; transgenic mice.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
NaV1.7 α1 subunit expression in wild-type (WT) and R192Q KI TG cultures. (A) Representative immunofluorescent examples of NaV1.7 α1 expression in WT and R192Q KI mouse trigeminal ganglia (TG) cultures. Nuclei are visualized with DAPI (blue); scale bar = 20 μm. Note extensive co-staining of NaV1.7 α1 (red) and β-tubulin III (green) protein in both WT and KI samples. (B) Western blot example showing protein expression of NaV1.7 α1 in TG from P12 mice. β-actin was used as a loading control. Histograms representing NaV1.7 α1 relative optical density values for each group (*p = 0.46, Mann-Whitney test; n = 8 experiments). Note the significant difference between WT and KI groups. (C) The histograms quantify the cell diameter distribution of NaV1.7 α1 immunofluorescence in different subgroups of WT and KI cultures (n = 3 experiments). The percentages of positive neurons for NaV1.7 were calculated by considering the total number of neurons labeled by β-Tubulin III as standard.
FIGURE 2
FIGURE 2
NaV1.7 α1 subunit expression in WT and R192Q KI TG cultures after pretreatment with ω-agatoxin IVA. (A) Representative immunofluorescent examples of NaV1.7 α1 expression in WT and R192Q KI mouse TG cultures. ω-agatoxin IVA (200 nM, overnight: in this and subsequent Figures the toxin is abbreviated as ω-Agatoxin) was used to specifically block CaV2.1 channels (that are mutated in the KI model). Nuclei are visualized with DAPI (blue); scale bar = 20 μm. Note extensive co-staining of NaV1.7 α1 (red) and β-tubulin III (green) protein in both WT and KI samples. (B) Histograms represent the percentage of NaV1.7-positive cells in WT and KI cultures. Note significant difference between WT and KI control groups (*p = 0.015, Mann–Whitney test; n = 7 experiments), and the decrease in NaV1.7 positive neurons in the KI after application of ω-agatoxin IVA (p = 0.035; n = 3 experiments). (C) The histograms quantify the cell diameter distribution of NaV1.7 α1 immunofluorescence in different subgroups in WT and KI ω-agatoxin IVA-treated cultures.
FIGURE 3
FIGURE 3
Co-expression of NaV1.7 α1 subunit and P2X3R in WT and R192Q KI cultures. (A) Representative examples of NaV1.7 α1–P2X3 immunostaining in WT and R192Q KI mouse TG cultures. Nuclei are visualized with DAPI (blue); scale bar = 20 μm. Note the extensive co-staining of NaV1.7 α1 (red) and P2X3 (green) protein in both WT and KI samples. (B) Histograms represent percentage co-expression of NaV1.7 α1–P2X3 proteins in WT and KI cultures (p = 0.95, Mann–Whitney test; n = 3 experiments). (C) The histograms quantify the cell diameter distribution of NaV1.7 α1–P2X3R immunofluorescence in different subgroups in WT and KI cultures.
FIGURE 4
FIGURE 4
NaV1.7 current in WT and R192Q KI TG neurons. (A) Mean amplitudes of the currents evoked by a square pulse 100 ms depolarizing step from –75 to –45 mV, in WT and R192Q KI TG neurons under control conditions and after application of 1 μM TTX. Note larger control current in KI (*p = 0.039, two-sample Student’s t-test). Number of cells in each group: n = 29 (WT, control), n = 20 (WT, TTX), n = 43 (KI, control), i = 24 (KI, TTX). (B) Representative traces of inward currents recorded from one WT neuron in response to the same stimulation in control and after 1 μM TTX; the trace of TTX-sensitive current was obtained by subtraction of the TTX-resistant current from the control. (C) Superimposed representative traces of WT and KI TTX-sensitive currents obtained by subtraction; note larger KI current. (D) Histograms represent the calculated mean amplitudes of the currents evoked by the same stimulus (100-ms step from –75 to –45 mV) in control and after Tp1a (7 nM, 30 min). *Indicates statistically significant change (two-sample Student’s t-test, p-values are given in the text body). Number of cells in each group: n = 29 (WT, control), n = 28 (WT, Tp1a), n = 43 (KI, control), n = 38 (KI, Tp1a). (E) Superimposed representative traces of WT and KI Tp1a-sensitive (NaV1.7) currents obtained by subtraction of the TTX-resistant current from the control; note larger NaV1.7 current in KI.
FIGURE 5
FIGURE 5
Effects of Tp1a on firing of TG neurons evoked by a 45-pA 300-ms square current pulse. (A) Cell-specific firing patterns recorded from three different WT TG neurons in response to the same stimulation. NS, no-spiking cell; SS, single-spike cell; MF, multiple-firing cell. (B) Distribution of firing patterns in WT and R192Q KI TG cultures is very similar in control and after application of Tp1a (7 nM, 30 min); chi-square test for proportions. Number of cells in each group for NS, SS, and MF cells, respectively: n (WT, control) = 15, 16, 109; n (WT, Tp1a) = 11, 18, 99; n (KI, control) = 16, 15, 125; n (KI, Tp1a) = 16, 21, 102. (C) Histograms represent the percent change of the number of spikes, generated by WT and KI neurons in control and after 30 min application of 7 nM Tp1a. *Indicates statistically significant change (Mann–Whitney test, p-values are given in the text body). Number of cells in each group: n (WT, control) = 109, n (WT, Tp1a) = 99, n (KI, control) = 133, n (KI, Tp1a) = 102. (D) Averaged firing threshold of WT and KI neurons in control and after Tp1a (7 nM, 30 min). *Indicates statistically significant change (two-sample Student’s t-test, p-values are given in the text body). Number of cells is as in (C). Note more negative KI threshold value in control and depolarized WT and KI thresholds after Tp1a application. (E) Representative traces of the first generated AP by WT and KI control and Tp1a-treated (7 nM, 30 min) neurons. Arrows indicate AP threshold.
FIGURE 6
FIGURE 6
Tp1a reduces rebound effect after a 300-ms hyperpolarizing pulse of 10 pA in R192Q KI TG neurons. (A) Representative responses of WT and R192Q KI neurons, recorded in control and after application of Tp1a (7 nM, 30 min). Arrow shows higher rebound in KI which is reduced by Tp1a. (B) Histograms represent rebound effect in WT and KI neurons under control conditions and after 30 min application of 7 nM Tp1a. *Indicates statistically significant change (two-sample Student’s t-test, p-values are given in the text body). Number of cells in each group: n = 90 (WT, control), n = 86 (WT, Tp1a), n = 109 (KI, control), n = 96 (KI, Tp1a). Note small but statistically significant difference between WT and KI neurons, and a reduction of the rebound effect in KI after Tp1a application.
FIGURE 7
FIGURE 7
OD1 elevates firing of TG neurons in response to a 45-pA 300-ms square current pulse. (A) Representative traces obtained from WT and R192Q KI TG neurons in control and after application of 10 nM OD1 (paired data, recordings from the same cell) or after application of 10 nM OD1 on cells pretreated with Tp1a (7 nM, 30 min; recordings from different cells). (B) OD1 application makes the AP threshold more negative and increases spike count both in WT and KI neurons (paired data); *Indicates statistically significant change (Mann-Whitney test, p-values are given in the text body). Number of cells in each group: n = 37 (WT, control), n = 36 (WT, OD1), i = 28 (KI, control), n = 25 (KI, OD1). The effect of OD1 is decreased when cells were pretreated with Tp1a (7 nM, 30 min); *Indicates statistically significant change (Mann–Whitney test, p-values are given in the text body).
FIGURE 8
FIGURE 8
Opposite effects of Tp1a and OD1 on spontaneous firing in TG neurons. (A) Diagram shows percentage of firing WT and R192Q KI TG neurons at different holding potentials in control and after Tp1a (7 nM, 30 min). *Indicates statistically significant change (Fisher test for proportions, p-values are given in the text body). Number of analyzed neurons at –70, –60, and –50 mV, respectively, are: n (WT, control) = 40, 40, 40; n (WT, Tp1a) = 32, 32, 31; n (KI, control) = 42, 42, 37; n (KI, Tp1a) = 33, 33, 33. (B) Bar charts represent average firing frequencies of WT and KI neurons in control and after Tp1a (7 nM, 30 min). *Indicates statistically significant change (paired Student’s t-test, p-values are given in the text body). Number of cells in each group: n = 31 (WT, control), n = 30 (WT, Tp1a), n = 23 (KI, control), n = 23 (KI, Tp1a). KI neurons fire with significantly higher frequency, which is reduced to the WT level after Tp1a application. (C) Application of 10 nM OD1 (paired data, recordings from the same cell) increases firing frequency of both WT and KI neurons at –50 mV. *Indicates statistically significant change (paired Student’s t-test, p-values are given in the text). Number of cells in each group: n = 17 (WT), n = 10 (KI), data are paired. (D) Examples of WT and KI neurons, firing at –50 mV before and after application of 10 nM OD1.
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