Figure S1 - Role of Ca2+ and L-Phe in Regulating Functional Cooperativity of Disease-Associated “Toggle” Calcium-Sensing Receptor Mutations

Functional studies of the CaSR double mutations in individual HEK293 cells. The panels show representative oscillation patterns from single cells. HEK-293 cells transfected with L173F/P221Q or L173P/P221L were loaded with Fura-2 AM for 15 min. [Ca2+]i was assessed by monitoring emission at 510 nm with excitation alternately at 340 or 380 nm as described in Methods. Each experiment began in Ca2+-free Ringer's buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, and 1.0 mM MgCl2, pH 7.4), followed by stepwise increases in [Ca2+]o until [Ca2+]i reached a plateau (up to 30 mM [Ca2+]o). a. Cells were transfected with pEGFP-N1-CaSR L173F/P221Q. b. Cells were transfected with pEGFP-N1-CaSR L173P/P221L. (DOCX)