TRPM channels modulate epileptic-like convulsions via systemic ion homeostasis

Abstract

Neuronal networks operate over a wide range of activity levels, with both neuronal and nonneuronal cells contributing to the balance of excitation and inhibition. Activity imbalance within neuronal networks underlies many neurological diseases, such as epilepsy. The Caenorhabditis elegans locomotor circuit operates via coordinated activity of cholinergic excitatory and GABAergic inhibitory transmission. We have previously shown that a gain-of-function mutation in a neuronal acetylcholine receptor, acr-2(gf), causes an epileptic-like convulsion behavior. Here we report that the behavioral and physiological effects of acr-2(gf) require the activity of the TRPM channel GTL-2 in nonneuronal tissues. Loss of gtl-2 function does not affect baseline synaptic transmission but can compensate for the excitation-inhibition imbalance caused by acr-2(gf). The compensatory effects of removing gtl-2 are counterbalanced by another TRPM channel, GTL-1, and can be recapitulated by acute treatment with divalent cation chelators, including those specific for Zn(2+). Together, these data reveal an important role for ion homeostasis in the balance of neuronal network activity and a novel function of nonneuronal TRPM channels in the fine-tuning of this network activity.

Figure 1. gtl-2 acts in non-neuronal tissues to modulate acr-2(gf) convulsions

(A) Images of wild type, acr-2(gf) and gtl-2(lf);acr-2(gf) locomotion. (B) Quantification of convulsion frequency in mutants of genotype as indicated. **, p<0.01, compared to acr-2(gf), ANOVA and Dunnett’s post hoc test. (C) Transgenic rescue of the suppression activities in gtl-2(0);acr-2(gf) and gtl-2(lf);acr-2(gf). ***, p<0.001, ANOVA and Bonferroni post hoc test. (D) Confocal image of anti-GFP immunostaining of GTL-2::GFP in an adult shows expression in the excretory cell (arrowhead) and epidermis (arrow). (E) Confocal image of anti-GFP immunostaining of GTL-2::GFP in a L4 larva expressing Pdpy-7:gtl-2cDNA::gfp. (F) Cell specific rescue of gtl-2 shows requirement in epidermis and excretory cell. Cell types are as follows: Pdpy-30 – all cells, Prgef-1 – pan-neuronal, Pmyo-3 – body muscles, Psulp-4 – excretory cell, and Pdpy-7 – epidermis. **, p<0.01, compared to gtl-2(lf);acr-2(gf), ANOVA and Dunnett’s post hoc test.

Figure 2. Physiology of gtl-2(lf) and its effects on acr-2(gf)

(A) Paralysis response to 500 µM aldicarb in gtl-2(lf) compared to wild type. (B) Representative traces and frequencies of endogenous acetylcholine and GABA postsynaptic currents from wild type (n=25), gtl-2(lf) (n=12), acr-2(gf) (n=14), and gtl-2(lf);acr-2(gf) (n=12) animals in 1 mM external CaCl₂ bath solution. Error bars indicate SEM. (C) Paralysis response to 200 µM aldicarb in wild type, acr-2(gf), and gtl-2(0);acr-2(gf) animals. *, p<0.05, compared to acr-2(gf), two-way ANOVA and Bonferroni post hoc tests. (D) Summary of frequencies of endogenous acetylcholine (left) and GABA (right) release in CaCl₂ bath solutions of 0.1 mM (wild type (n=14), acr-2(gf) (n=9), and gtl-2(lf);acr-2(gf) (n=10)), 0.5 mM (wild type (n=12), acr-2(gf) (n=14), and gtl-2(lf);acr-2(gf) (n=11)), and 1 mM (wild type (n=25), acr-2(gf) (n=14), and gtl-2(lf);acr-2(gf) (n=12)). Error bars indicate SEM. Statistics in B, D used SigmaStat 3.5 (Aspire Software International): *, p<0.05, ***, p<0.001 by Student’s t-test, or the Mann-Whitney rank sum test for GABA data in 0.5 mM CaCl₂ according to the normality of datum distribution for data.

Figure 3. gtl-2 specifically influences the overexcitation phenotype in acr-2(gf), but not of goa-1(lf) mutants with increased cholinergic transmission

(A) Quantification of convulsion frequency in mutants of genotypes indicated. -, no mutation; x, has mutation. ***, p<0.001, compared to gtl-2(lf);acr-2(gf), ANOVA and Bonferroni post hoc test. (B) Images of animals of genotype indicated. gtl-2(lf) does not alter locomotion behavior of goa-1(ep275) animals. (C) Paralysis response to 200 µM aldicarb in animals of genotypes indicated. n=3 trials of 10 animals per genotype.

Figure 4. Ion homeostasis plays a key role in the acr-2(gf) phenotype

(A) Quantification of convulsion frequency of acr-2(gf) and gtl-2(lf);acr-2(gf) with genetic mutations in gon-2 and gtl-1 or treated with gtl-1 RNAi to reduce the function of different TRPM channels. *** = p<0.001, ANOVA and Bonferroni post hoc test. (B) Images of acr-2(gf) animals soaked in either M9 (control), EDTA or TPEN. (C) Convulsion frequency of acr-2(gf) and gtl-2(lf);acr-2(gf) soaked in the cation chelators EDTA (75 mM), EGTA (75 mM), DTPA (75mM), and TPEN (100 µM). Statistics in A, C, *** = p<0.001, ANOVA and Bonferroni post hoc test. (D) Model of the regulation of locomotor circuit by ion homeostasis and the three TRPM channels. GON-2 and GTL-1 act in the intestine (I) to allow divalent cation influx, whereas GTL-2 acts in the excretory cell (EC) for cation efflux [9](and this study). Neuronal activity (MN) likely influences local cation levels (dark blue oval). Our data suggest that both the systemic cation fluctuations due to the function of the three TRPM channels as well as local ion fluctuations involving GTL-2 in the epidermis (E) modulate the excitability of the locomotor circuit, hence contractions of muscles (M).