Ca2+ Regulation of Cav3.3 T-type Ca2+ Channel Is Mediated by Calmodulin

Abstract

Calcium-dependent inactivation of high voltage-activated Ca²⁺ channels plays a crucial role in limiting rises in intracellular calcium (Ca²⁺_i). A key mediator of these effects is calmodulin, which has been found to bind the C-terminus of the pore-forming α subunit. In contrast, little is known about how Ca²⁺_i can regulate low voltage-activated T-type Ca²⁺ channels. Using whole cell patch clamp, we examined the biophysical properties of Ca²⁺ current through the three T-type Ca²⁺ channel isoforms, Ca_v3.1, Ca_v3.2, or Ca_v3.3, comparing internal solutions containing 27 nM and l μM free Ca²⁺ Both activation and inactivation kinetics of Ca_v3.3 current in l μM Ca²⁺_i solution were more rapid than those in 27 nM Ca²⁺_i solution. In addition, both activation and steady-state inactivation curves of Ca_v3.3 were negatively shifted in the higher Ca²⁺_i solution. In contrast, the biophysical properties of Ca_v3.1 and Ca_v3.2 isoforms were not significantly different between the two internal solutions. Overexpression of CaM₁₂₃₄ (a calmodulin mutant that doesn't bind Ca²⁺) occluded the effects of l μM Ca²⁺_i on Ca_v3.3, implying that CaM is involved in the Ca²⁺_i regulation effects on Ca_v3.3. Yeast two-hybrid screening and co-immunoprecipitation experiments revealed a direct interaction of CaM with the carboxyl terminus of Ca_v3.3. Taken together, our results suggest that Ca_v3.3 T-type channel is potently regulated by Ca²⁺_i via interaction of Ca²⁺/CaM with the carboxyl terminus of Ca_v3.3.