tmc-1 encodes a sodium-sensitive channel required for salt chemosensation in C. elegans

Abstract

Transmembrane channel-like (TMC) genes encode a broadly conserved family of multipass integral membrane proteins in animals. Human TMC1 and TMC2 genes are linked to human deafness and required for hair-cell mechanotransduction; however, the molecular functions of these and other TMC proteins have not been determined. Here we show that the Caenorhabditis elegans tmc-1 gene encodes a sodium sensor that functions specifically in salt taste chemosensation. tmc-1 is expressed in the ASH polymodal avoidance neurons, where it is required for salt-evoked neuronal activity and behavioural avoidance of high concentrations of NaCl. However, tmc-1 has no effect on responses to other stimuli sensed by the ASH neurons including high osmolarity and chemical repellents, indicating a specific role in salt sensation. When expressed in mammalian cell culture, C. elegans TMC-1 generates a predominantly cationic conductance activated by high extracellular sodium but not by other cations or uncharged small molecules. Thus, TMC-1 is both necessary for salt sensation in vivo and sufficient to generate a sodium-sensitive channel in vitro, identifying it as a probable ionotropic sensory receptor.

Figure 1. Expression of TMC-1 in chemosensory neurons. A-B

Expression of the tmc-1::mCherry promoter fusion in the ASH neurons. Animals expressing the extrachromosomal array ljEx470[tmc-1::mCherry] as well as ljEx239[sra-6::YC3.60] which specifically labels the ASH neurons. Panel A shows a red fluorescence image showing the expression pattern of the tmc-1::mCherry reporter; the ASH neuron is indicated by the arrow. Panel B is a DIC/fluorescence image of the same animal showing colabelling of tmc-1::mCherry with sra-6::YC3.60 in ASH Expression was also seen in the following neurons: ADF, ASE, PHA, ADL, CEP, NSM, and at lower frequency, AWC, AFD and BAG (see also Supplemental Figures 1 and 2). C. Localization of a tmc-1::mCherry protein fusion in the ASH neurons. Shown is a DIC/fluorescence image of a strain carrying a transgenic array (ljEx483[gpa-11::tmc-1::mCherry]) in which the tmc-1 cDNA is fused in frame to mCherry and expressed in ASH under the gpa-11 promoter. The ASH sensory cilia (thin arrows) and cell body (thick arrow) are indicated.

Figure 2. tmc-1 is specifically required for high salt avoidance behaviour. A

Effect of tmc-1 on 250mM NaCl avoidance. For each genotype at least 370 animals were tested in population drop assays. Avoidance index (A.I.) indicates the fraction of animals reversing following stimulus application; error bars for these and other panels indicates SEM. One-way ANOVA with Bonferroni correction was used to test significance. tmc-1(ok1859) animals were significantly different from wild-type (p<0.0005), while all tmc-1 rescue lines were significantly different from tmc-1(ok1859) (p<0.0005), but not from wild-type. B. Dose response for NaCl avoidance. For each data point, at least 170 animals were tested. At all concentrations tmc-1(ok1859) were significantly different from wild-type (p<0.005) and all tmc-1 rescue lines were significantly different from tmc-1(ok1859) (p<0.05). C. Effect of tmc-1 on other ASH-dependent escape behaviours. For each genotype at least 260 animals were tested for nose touch and at least 320 for CuCl₂ avoidance. No significant difference was observed between wild type and tmc-1(ok1859). D. Dose response for glycerol avoidance. For each data point, at least 70 animals were tested. No significant difference was detected between wild-type and tmc-1(ok1859) across all concentrations. E. Dose response for Na gluconate avoidance. For each data point, at least 70 animals were tested (except ablated animals, for which a minimum of 25 animals were tested). tmc-1(ok1859) was significantly different from wild-type across all concentrations (p<0.05). The tmc-1; gpa-11::tmc-1 rescue line was significantly different from tmc-1(ok1859) (p<0.05). F. Chemotaxis behaviour of wild-type, tmc-1 mutant and animals with heterologous tmc-1 expression in ASK to Na gluconate. At least 790 animals were assayed for each genotype. Wild type was significantly different from tmc-1(ok1859) (p<0.05) and tmc-1; srbc-64::tmc-1 was significantly different (p<0.05) from tmc-1(ok1859).

Figure 3. tmc-1 is specifically required for ASH chemosensory responses to salt

A. ASH calcium responses to external salt stimulation. Shown are averaged traces of ASH calcium transients in wild-type, tmc-1 mutant and ASH-rescued animals in response to a 10s NaCl concentration upstep of 0-500 mM. Traces indicate average percent ΔR/R₀ where R is the fluorescence emission ratio and R₀ the baseline ratio; the gray band represents SEM. Statistical significance for all experiments was evaluated by one-way ANOVA with Bonferroni correction. The time of the NaCl upstep is shown in blue; n≥13. B. Quantification of ASH calcium responses in mutant and rescued animals. 17<n<51 animals were tested for each data point; error bars for this and other panels indicate SEM. For all concentrations ≥100 mM, the response of tmc-1 was statistically different from wild-type and all rescue lines (p<.05). C. ASH calcium responses to sodium gluconate. 5<n<19 animals were tested for each data point. For all concentrations ≥100 mM, the response of tmc-1 was statistically different from wild-type and the rescue line (p<.05). D. ASH calcium responses to MgCl₂. 4<n<12 animals were tested for each data point. For all concentrations the response of tmc-1 was not statistically different from wild-type. E. ASH calcium responses to glycerol. 5<n<38 animals were tested for each data point. For all concentrations the response of tmc-1 was not statistically different from wild-type. F. ASK calcium responses to sodium gluconate. Shown are averaged R/R₀ traces in 12 wild-type, and 15 srbc-64::tmc-1 animals, which express TMC-1 heterologously in ASK, in response to a 10 second stimulation with 0.5 M sodium gluconate. G. Quantification of ASK calcium responses to sodium gluconate. 7<n<24 animals were tested for each data point. For all concentrations ≥100mM, the response of srbc-64::tmc-1 was statistically different from wild-type (p<.05).

Figure 4. Sodium-sensitive cation currents in TMC1-expressing cells. A

Inward currents were elicited in TMC-1-expressing CHO-K1 cells by external addition of 150 mM NaCl, but not by 300mM mannitol or glucose, in whole-cell voltage clamp recordings at -60 mV (n=7) using native chloride conditions (see methods). Unless otherwise indicated (i.e. panels B and E) the external buffer contained 140 mM NaCl. When GFP was co-transfected, 76% of GFP-positive cells showed the inward current reponses; mock-transfected or untransfected cells lacked such responses (Supplemental Figure 8D). Inset: Representative current-voltage relationship of NaCl-induced inward currents in TMC-1-expressing CHO-K1 cells. B. NaCl dose-response for inward currents of TMC-1-expressing cells. Filled circles represent mean responses to extracellular NaCl (n=9-17 for each point); error bars for this and other panels indicates SEM. Fitting to the Hill equation gives a Hill coefficient of 4.5 and EC50 of 220 mM. C. Activation of currents in TMC-1 CHO-K1 cells by other salts. Shown are mean current responses at -60 mV upon addition of 300 mM of the indicated salt to the recording buffer (n=6-11). D. Intracellular Ca²⁺ levels were elevated in TMC-1 CHO-K1 cells in response to addition of external 150 mM NaCl but not to 300 mM mannitol (n=34). Mock-transfected or untransfected cells lacked such responses, as did transfected cells in Ca²⁺-free external buffer (Supplemental Figure 9). E. Relative cation permeabilities of TMC-1 calculated from the reversal potentials of basal currents with CsCl in the internal solution and the indicated chloride salt in the external solution. Error bars indicate SEM. Inset: representative current-voltage relationships for different external salts in TMC-1-expressing CHO-K1 cells. F. 1 mM GdCl₃ blocked NaCl-induced inward currents in TMC-1-expressing CHO-K1 cells (n=11).