Figure 1.

Functional analysis of CaM mutants and Ca²⁺-dependent binding. (A) Representative currents recorded from HEK293 cells coexpressing with TRPC6 and CaM_WT or a CaM mutant (CaM₁₂, CaM₃₄, or CaM₁₂₃₄). Muscarinic receptor (M₁R) is also coexpressed for the electrophysiologic experiments in HEK293 cells. The inset shows typical I–V curve of TRPC6. (B) Fractions of residual currents plotted as a function of time after the peak. Black circles indicate control data from TRPC6 with M₁R-transfected cells only (n=14, CaM_endo), here and throughout. (C) Representative profiles of Ca²⁺-dependent binding of CaMs to the TRPC6 CBD. FRET measurement is explained in the Methods section. (D) Quantification of the affinity of binding of CBD_WT to CaM mutants by plotting donor free ([D]_free) concentrations against FRET signal (FR). Data from various cells were collected at their maximum FR values in (C). Lines represent fitting of CBD_WT versus CaM_MUT. FRET data are summarized in Supplemental Table 1.