Figure 2.

Ca²⁺ acts directly at the plasma membrane via electrostatic mechanisms. (A–D) Freshly prepared membrane sheets were treated in the presence (A, B) or absence (C, D) of magnesium for 10 min at 37°C with a defined Ca²⁺ concentration, fixed, immunostained for membrane proteins as indicated (Syp, TfR and Syx1 refer to synaptophysin, transferrin receptor and syntaxin 1 respectively) and analysed as in Figure 1. (A) Magnified views from immunostained membrane sheets after treatments with no (upper panels) or 54 μM (lower panels) Ca²⁺. (B–D) Change in immunostaining intensity induced by 54 μM (B), 850 nM (C) or 225 nM (D) Ca²⁺. Residual fluorescence intensities were normalized to the intensities obtained in the absence of Ca²⁺ and expressed as percentage. Values are means±s.e.m. (n=3–6 independent experiments; 18–106 membrane sheets were analysed for each condition in one experiment). (E) At pH 7.4, the amino acids Asp (D) and Glu (E) are negatively charged whereas Arg (R) and Lys (K) carry positive charges. For each membrane protein, the number of the negatively and positively charged amino acids within the cytoplasmic regions was determined and %DE–%RK plotted against fluorescence remaining after Ca²⁺ treatment with 850 nM (black) or 54 μM Ca²⁺ (grey).