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 (Ca2+i). A key mediator of these effects is calmodulin, which has been found to bind the C-terminus of the pore-forming αsubunit. In contrast, little is known about how Ca2+ican 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, Cav3.1, Cav3.2, or Cav3.3, comparing internal solutions containing 27 nM and l μM free Ca2+. Both activation and inactivation kinetics of Cav3.3 current in l μM Ca2+isolution were more rapid than those in 27 nM Ca2+isolution. In addition, both activation and steady-state inactivation curves of Cav3.3 were negatively shifted in the higher Ca2+isolution. In contrast, the biophysical properties of Cav3.1 and Cav3.2 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+ion Cav3.3, implying that CaM is involved in the Ca2+iregulation effects on Cav3.3. Yeast two-hybrid screening and co-immunoprecipitation experiments revealed a direct interaction of CaM with the carboxyl terminus of Cav3.3. Taken together, our results suggest that Cav3.3 T-type channel is potently regulated by Ca2+ivia interaction of Ca2+/CaM with the carboxyl terminus of Cav3.3.