[125I]calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and arrhenius analysis

Binding of ¹²⁵I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca²⁺-dependent membrane binding of ¹²⁵I]calmodulin was found to have a B_max of 284 pmol/mg protein and an apparent affinity with a K_d of 131 nM. Kinetic analysis indicates that at 37°C, the dissociation of ¹²⁵I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7×10^–1 min^–1 and a half-time (t_1/2) of 1.8 min for the fast component, and a k of 4.8×10^–2 min^–1 and a t_1/2 of 14.5 min for the slow component. At 0°C, substantial dissociation still occurred, with a k of 4.5×10^–2 min^–1 and a t_1/2 of 15.3 min for the fast component, and a k of 5.5×10^–3 min^–1 and a t_1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associated and dissociate calmodulin. In Arrhenius analysis of temperature effects, ¹²⁵I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (T_d) estimated to be 23.8°C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of ¹²⁵I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10^–7–10^–6 M), a steroid hormone, and decreased by ethanol (50–200 mM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.Abbreviations used CaM calmodulin - SPM synaptic plasma membrane - ATPase adenosine triphosphatase - Tris tris(hydroxymethyl)aminomethane - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - SDS sodium dodecyl sulfate - TFP trifluoperazine - K_d dissociation constant - B_max maximum binding - k first-order rate constant - t_1/2 half-time - T_d transition temperature