Figure 3

Mutational effects of pair residues on the intrinsic equilibrium (K_int) of bidirectional Ca²⁺ movements (Ca²⁺/Ca²⁺ exchange).

(A) Topological positions of pair residues within the ion pocket of NCX_Mj. Matching pair residues are marked with the same color. (B) The K_m and V_max values of the Na⁺/Ca²⁺ and Ca²⁺/Ca²⁺ exchange reactions were measured by varying the concentrations of ⁴⁵Ca²⁺ (20–2000 μM) in the assay medium at fixed (saturating) concentrations of intravesicular Na⁺ (160 mM) or Ca²⁺ (250 μM), as described in Methods (see also Figure S1). The bars represent the mean ± SE. (C) The K_m values of the Ca²⁺/Ca²⁺ exchange reaction were measured at the ‘extracellular’ (K″_m) and ‘cytosolic’ (K′_m) sides by varying the Ca²⁺ concentrations in one compartment and keeping the fixed (saturating) concentrations of Ca²⁺ in the trans compartment. (D) The K_int values were calculated as the K′_m/K″_m ratio and plotted vs the k_cat values for the same mutant. The k_cat values were calculated as k_cat = V_max/[E]_t. The expression levels of [E]_t were normalized by using the GFP assay (see Methods).