Ca Regulation of Ca3.3 T-type Ca Channel Is Mediated by Calmodulin.

Calcium-dependent inactivation of high voltage-activated Ca channels plays a crucial role in limiting rises in intracellular calcium (Ca). A key mediator of these effects is calmodulin, which has been found to bind the C-terminus of the pore-forming subunit. In contrast, little is known about how Ca can regulate low voltage-activated T-type Ca channels. Using whole cell patch clamp, we examined the biophysical properties of Ca current through the three T-type Ca channel isoforms, Ca3.1, Ca3.2, or Ca3.3, comparing internal solutions containing 27 nM and l μM free Ca Both activation and inactivation kinetics of Ca3.3 current in l μM Ca solution were more rapid than those in 27 nM Ca solution. In addition, both activation and steady-state inactivation curves of Ca3.3 were negatively shifted in the higher Ca solution. In contrast, the biophysical properties of Ca3.1 and Ca3.2 isoforms were not significantly different between the two internal solutions. Overexpression of CaM (a calmodulin mutant that doesn't bind Ca) occluded the effects of l μM Ca on Ca3.3, implying that CaM is involved in the Ca regulation effects on Ca3.3. Yeast two-hybrid screening and co-immunoprecipitation experiments revealed a direct interaction of CaM with the carboxyl terminus of Ca3.3. Taken together, our results suggest that Ca3.3 T-type channel is potently regulated by Ca via interaction of Ca/CaM with the carboxyl terminus of Ca3.3.