Figure 5.
Effects of mutant calmodulins on the Ca sensitivity of Cl channels. (A) Current and fluorescence recordings from two cells that displayed similar increments of intracellular Ca in response to a flash series. The red traces were recorded from a cell transfected with the mutant CaM R2, in which one of the two NH2-terminal Ca binding sites is disabled by a point mutation. Note that more flashes (i.e., higher Ca concentrations) are necessary to achieve half-maximal activation in the cell expressing the mutant. Normalized recordings obtained at −40 mV. (B) Dose–response relations derived from the data in A. Transfection with CaM R2 shifted the dose–response curve to the right, increasing K1/2 from 1.07 to 2.09 at constant Hill coefficient (n = 2.3). (C) Collected results of transfection experiments with mutant calmodulins. The disabled Ca binding sites are indicated by dark gray dots on the CaM symbols. Asterisks indicate the significance level (nonpaired Student's t test) compared with control cells. “Exp.” denotes the number of cells examined (two to seven transfections each). Mutations in the Ca binding sites 1, 2, and 4 caused a reduction of Ca sensitivity, while a mutation in binding site 3 was ineffective. The totally inactive mutant CaM R1234 points to a requirement of Ca for the association of CaM with the Cl channel (see Discussion). Ranges for K1/2 values were (in μM) as follows. Control, 0.91–1.98; CaM WT, 0.90–2.07; CaM R1, 1.33–2.17; CaM R2, 1.06–2.40; CaM R3, 0.83–1.74; CaM R4, 1.16–2.13; CaM R12, 1.06–2.49; CaM R34, 1.19–1.90; CaM R1234, 0.63–2.05.