Calcium–dependent binding of calmodulin to retinal gap junction proteins

Abstract

Abstract: : Purpose: Changes in intracellular Ca²⁺ regulate the activity of many enzymes and channels, frequently through binding interactions with Ca²⁺–binding proteins such as calmodulin. Changes in intracellular Ca²⁺ have been observed to influence gap junctional coupling in many cells, including neurons. The present study examines mechanisms by which Ca²⁺ and calmodulin may regulate gap junctions in the retina. Methods: Intracellular domains of the retinal gap junction proteins connexin35 (Cx35) and Cx34.7 were expressed as GST fusion proteins in bacteria and yeast. We used surface plasmon resonance to evaluate calmodulin binding to the connexin intracellular domains. Cx35 and Cx34.7 intracellular loop (I–loop) and carboxyl terminal (CT) domain GST fusion proteins were captured with an anti–GST antibody immobilized on a CM5 chip. Binding was analyzed with a Biacore 2000 SPR instrument. Results: Calmodulin was found to bind specifically to the C–terminal domains of both Cx35 and Cx34.7. No binding was observed to the I–loops or to GST control proteins. Calmodulin binding was concentration–dependent and was dependent on the presence of Ca²⁺. Cx35CT bound calmodulin with a Kd of 0.60 µM at 1 mM free Ca²⁺, while Cx34.7CT bound with a Kd of 0.26 µM. The association rates for both Cx35CT and Cx34.7CT were too fast to measure accurately, but dissociation was first order. Following binding to 1 µM calmodulin at 1 mM free Ca²⁺, the dissociation rate constants were 3.3 x 10^–3 s^–1 for Cx35CT and 6.4 x 10^–3 s^–1 for Cx34.7CT. With these off rates, nearly all bound calmodulin dissociates within a few minutes of removal of calmodulin from the solution, even in the presence of Ca²⁺. Conclusions: Calmodulin binds to two retinal neuronal connexins, Cx35 and Cx34.7, with approximately 2–fold different Kd’s. The binding is domain–specific, Ca²⁺–dependent and rapid. The low micromolar Kd’s and rapid on and off rates suggest that this binding interaction may change dynamically in neurons, where free calmodulin concentrations are a few micromolar. This binding interaction may be related to the Ca²⁺–dependent changes in gap–junctional coupling that have been observed in neurons, and may be a mechanism by which neurons that express these connexins, such as cone photoreceptors, regulate their coupling.

Keywords: gap junctions/coupling • calcium • cell–cell communication