Mutation of the S3–S4 linker eliminates Mg²⁺ response. (A) Diagram illustrating the design of mutation. (B) Representative Ca²⁺ imaging trace. (C) Summary of the amplitude ratio between Mg²⁺- and capsaicin-induced fluorescence responses for wild type (black bar; n = 48) and Rs₃s₄ (blue bar; n = 65). (D) Representative current trace recorded at +80 mV in response to 130 mM Mg²⁺ and 3 µM capsaicin. (E) Summary of amplitude ratio between Mg²⁺- and capsaicin-induced current for wild type (black bar; n = 5) and Rs₃s₄ (blue bar; n = 4). ***, P < 0.001.