Fig. 3. Dissociation of Ca²⁺ from the complex between BSCaM_IQ and CaM or N_xCCaM.

The fluorescent Ca²⁺ chelator quin-2 was used as a Ca²⁺ trap. Dissociation of Ca²⁺ from native or mutant CaM therefore corresponds directly with a Ca²⁺-dependent increase in quin-2 fluorescence. A range of quin-2 concentrations was investigated to ensure that a concentration sufficient to prevent rebinding of Ca²⁺ was used. (A) Dissociation of Ca²⁺ from free CaM (lower trace) and from the CaM-BSCaM_IQ complex (upper trace). The quin-2 fluorescence time course for Ca²⁺ dissociation from free CaM was generated by mixing a solution containing 4 µM CaM and 50 µM CaCl₂ with an equal volume of 400 µM quin-2. These data are fit by a single exponential with a rate constant of 10.3±0.7 s⁻¹ and an amplitude corresponding with dissociation of ~2 Ca²⁺ ions. Dissociation of two additional Ca²⁺ ions occurred within the instrument dead time. The quin-2 fluorescence time course for Ca²⁺ dissociation from the CaM-BSCaM_IQ complex was generated by mixing a solution containing 25 µM BSCaM_IQ, 4 µM CaM and 50 µM CaCl₂ with an equal volume of 400 µM quin-2. The data presented are fit by a double exponential. The slower process has an apparent rate constant of 14.7±0.5 s⁻¹ and an amplitude corresponding with dissociation of ~2 Ca²⁺ ions. The faster rate was derived from data measured over a shorter time interval (inset). These data are fit by a double exponential with rate constants of 427.4±35 and 11.7±0.5 s⁻¹, and respective amplitudes corresponding with dissociation of ~1.5 and ~2 Ca²⁺ ions. If the faster process is forced to have an amplitude corresponding with dissociation of 2 Ca²⁺ ions, then apparent rate constants of 473.4±37.9 s⁻¹ and 12.9±0.7 s⁻¹ are derived for the faster and slower rates. (B) Dissociation of Ca²⁺ from the N_xCCaM-BSCaM_IQ complex (●) and from free N_xCCaM (○). For these experiments a solution containing 4 µM N_xCCaM and 50 µM CaCl₂ in the presence or absence of 25 µM BSCaM_IQ was mixed with an equal volume of 400 µM quin-2. The two resulting quin-2 fluorescence time courses are essentially superimposable. Ca²⁺-dissociation rate constants of 10.8±0.6 s⁻¹ and 11.8±0.5 s⁻¹ were derived for free and bound N_xCCaM from fits of these data to single exponentials with amplitudes corresponding to dissociation of ~2 Ca²⁺ ions.