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 a 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, Cav3.1, Cav3.2, or Cav3.3, comparing internal solutions containing 27 nMand lmMfree Ca2+. Both activation and inactivation kinetics of Cav3.3 current in l mM Ca2+ i solution were more rapid than those in 27 nM Ca2+ i solution. In addition, both activation and steady-state inactivation curves of Cav3.3were negatively shifted in the higherCa2+ i solution. 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 mMCa2+ i on Cav3.3, implying that CaM is involved in the Ca2+ i regulation 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+ i via interaction of Ca2+/CaM with the carboxyl terminus of Cav3.3.