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

Calcium-dependent inactivation of high voltage-activated Ca2+ channels plays a crucial role in limiting rises in intracellular calcium (Ca-i(2+)). A key mediator of these effects is calmodulin, which has been found to bind the C-terminus of the pore-forming a subunit. In contrast, little is known about how Ca-i(2+) 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(v)3.1, Ca(v)3.2, or Ca(v)3.3, comparing internal solutions containing 27 nM and l mu M free Ca2+. Both activation and inactivation kinetics of Ca(v)3.3 current in l mu M Ca-i(2+) solution were more rapid than those in 27 nM Ca-i(2+) solution. In addition, both activation and steady-state inactivation curves of Ca(v)3.3 were negatively shifted in the higher Ca-i(2+) solution. In contrast, the biophysical properties of Ca(v)3.1 and Ca(v)3.2 isoforms were not significantly different between the two internal solutions. Overexpression of CaM1234 (a calmodulin mutant that doesnt bind Ca2+) occluded the effects of l mu M Ca-i(2+) on Ca(v)3.3, implying that CaM is involved in the Ca-i(2+) regulation effects on Ca(v)3.3. Yeast two-hybrid screening and co-immunoprecipitation experiments revealed a direct interaction of CaM with the carboxyl terminus of Ca(v)3.3. Taken together, our results suggest that Ca(v)3.3 T-type channel is potently regulated by Ca-i(2+) via interaction of Ca2+/CaM with the carboxyl terminus of Ca(v)3.3.