The DEG/ENaC protein MEC-10 regulates the transduction channel complex in Caenorhabditis elegans touch receptor neurons

Abstract

Gentle touch sensation in Caenorhabditis elegans is mediated by the MEC-4/MEC-10 channel complex, which is expressed exclusively in six touch receptor neurons (TRNs). The complex contains two pore-forming subunits, MEC-4 and MEC-10, as well as the accessory subunits MEC-2, MEC-6, and UNC-24. MEC-4 is essential for channel function, but beyond its role as a pore-forming subunit, the functional contribution of MEC-10 to the channel complex and to touch sensation is unclear. We addressed this question using behavioral assays, in vivo electrophysiological recordings from TRNs, and heterologous expression of mutant MEC-10 isoforms. Animals with a deletion in mec-10 showed only a partial loss of touch sensitivity and a modest decrease in the size of the mechanoreceptor current (MRC). In contrast, five previously identified mec-10 alleles acted as recessive gain-of-function alleles that resulted in complete touch insensitivity. Each of these alleles produced a substantial decrease in MRC size and a shift in the reversal potential in vivo. The latter finding indicates that these mec-10 mutations alter the ionic selectivity of the transduction channel in vivo. All mec-10 mutant animals had properly localized channel complexes, indicating that the loss of MRCs was not attributable to a dramatic mislocalization of transduction channels. Finally, electrophysiological examination of heterologously expressed complexes suggests that mutant MEC-10 proteins may affect channel current via MEC-2.

Figure 1.

mec-10 mutant alleles. A, Touch sensitivity of mec-10 animals. Each animal was touched 10 times, as described in Materials and Methods (mean ± SEM, n = 30 for each). B, Location of missense mutations in mec-10 mutant alleles within conserved regions of DEG/ENaC proteins. Protein sequences were aligned using COBALT (Papadopoulos and Agarwala, 2007). Except ASIC1a (Gallus gallus), all sequences are from C. elegans. Gray shading indicates conserved residues at sites mutated in mec-10. Residues conserved among all the proteins are highlighted in blue. Transmembrane domains are represented by black boxes. C, The ok1104 allele carries a 143 bp deletion (black bar) at the junction of the fourth intron and the fifth exon in the mec-10 gene. Location of primer pairs used for RT-PCR amplification is indicated in gray (F, R1, and R2). D, Putative protein products in ok1104 animals as derived from conceptual translation of three different RT-PCR products (see Results). Transmembrane domains are represented by black boxes, foreign amino acid residues by red bars, and a deletion by a white bar.

Figure 2.

Effects of mec-10 RNAi. A, RNAi against mec-10 in lin-35(n745) has no effect on animals with wild-type mec-10 or animals with the ok1104 deletion allele and enhances the touch sensitivity of animals with missense mutations in mec-10. Animals were fed bacteria expressing dsRNA against either GFP (white bars) or mec-10 (black bars) and assayed for touch response as described in Materials and Methods. Bars are mean ± SEM for n = 41–81 animals. Significance in all panels from Student's t test: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.0001. Numbers in parentheses represent the number of animals that were completely unresponsive. B, RNAi against mec-10 results in a partial loss of touch sensitivity in a strain that is hypersensitive to RNAi [P_unc-119sid-1 P_unc-119gfp P_mec-6mec-6; lin-15b(n744)], whereas RNAi against mec-4 produces a more dramatic loss of touch sensitivity. Animals were fed bacteria expressing dsRNA against either GFP (control), mec-10, or mec-4 as indicated and assayed for touch response as described in Materials and Methods (mean ± SEM, n = 60 for each). C, RNAi against mec-10 in a hypersensitive RNAi background (P_unc-119sid-1 P_unc-119gfp P_mec-6mec-6) reduces the touch sensitivity of animals with wild-type mec-10 (control) and does not affect the touch response of animals with a deletion in mec-10 (ok1104 or tm1552). Animals were fed bacteria expressing dsRNA against either GFP (white bars) or mec-10 (black bars) and assayed for touch response as described in Materials and Methods (mean ± SEM, n = 90–100 for each).

Figure 3.

mec-10 and mec-4 mutants have MRCs with decreased amplitude. A, Examples of in vivo recordings of MRCs from PLM touch receptor neurons in wild-type, mec-10, and mec-4 animals (at −74 mV) in response to a saturating mechanical stimulus (top). Each trace is an average of 20 sweeps. Stimulus pressure is indicated above each trace. B, Maximum peak amplitude of MRC recorded from PLM touch receptor neurons (at −74 mV), at the onset (black bars) and offset (white bars) of a response to a saturating mechanical stimulus (mean ± SEM, numbers in parentheses indicate number of cells tested).

Figure 4.

Point mutations in mec-10 and mec-4 alter the reversal potential of the MRC, whereas a deletion in mec-10 does not affect the reversal potential. Current–voltage relations for MRCs recorded from PLM cells at the onset of a saturating mechanical stimulus in mutant (gray) and wild-type (black) animals (mean ± SEM). MRCs were recorded at −114 to +66 mV in 20 mV increments. Current was normalized (I_norm) to the current recorded at a holding potential of −114 mV, and membrane potential (V_m) was adjusted for voltage attenuation as described in Materials and Methods. A, mec-10(ok1104) (n = 4) compared with wild-type (n = 7). B, mec-10(u390) (n = 3), mec-10(u332) (n = 4), and mec-10(e1715) (n = 4) compared with wild-type (n = 7). C, mec-10(e1515) (n = 3) and mec-4(u339) (n = 3) compared with wild type (n = 7).

Figure 5.

MEC-10 mutant proteins inhibit the amiloride-sensitive current in Xenopus oocytes in a MEC-2-dependent manner. Amiloride-sensitive current (I_amil) recorded at −85 mV from the MEC-4d channel complex expressed with wild-type and mutant forms of MEC-10 as indicated beneath each bar (mean ± SEM, numbers in parentheses represent number of cells tested). A, Oocytes expressing MEC-4d, MEC-2, MEC-6, and wild-type and mutant forms of MEC-10 as noted. Except for the G680E variant, currents recorded from channels with MEC-10 variants were significantly different from currents from channels with wild-type MEC-10 (p < 0.00001, Student's t test). B, Oocytes expressing MEC-4d, MEC-2, and wild-type and mutant forms of MEC-10 as noted. Currents recorded from channels with MEC-10 variant proteins were significantly different from those from channels with wild-type MEC-10 (p < 0.00001, Student's t test). C, Oocytes expressing MEC-4d, MEC-6, and wild-type and mutant forms of MEC-10 as noted. Currents recorded from channels with G680E were significantly larger than currents from channels with wild-type MEC-10 (p < 0.05, Student's t test). No significant difference in current between channels with any of the other MEC-10 variants and those with wild-type MEC-10 (p = 0.12–0.97, Student's t test).