Cloning and Expression of the Human T-Type Channel Cav3.3: Insights into Prepulse Facilitation

Abstract

The full-length human Cav3.3 (α1I) T-type channel was cloned, and found to be longer than previously reported. Comparison of the cDNA sequence to the human genomic sequence indicates the presence of an additional 4-kb exon that adds 214 amino acids to the carboxyl terminus and encodes the 3′ untranslated region. The electrophysiological properties of the full-length channel were studied after transient transfection into 293 human embryonic kidney cells using 5mM Ca2+ as charge carrier. From a holding potential of −100mV, step depolarizations elicited inward currents with an apparent threshold of −70mV, a peak of −30mV, and reversed at +40mV. The kinetics of channel activation, inactivation, deactivation, and recovery from inactivation were very similar to those reported previously for rat Cav3.3. Similar voltage-dependent gating and kinetics were found for truncated versions of human Cav3.3, which lack either 118 or 288 of the 490 amino acids that compose the carboxyl terminus. A major difference between these constructs was that the full-length isoform generated twofold more current. These results suggest that sequences in the distal portion of Cav3.3 play a role in channel expression. Studies on the voltage-dependence of activation revealed that a fraction of channels did not gate as low voltage-activated channels, requiring stronger depolarizations to open. A strong depolarizing prepulse (+100mV, 200ms) increased the fraction of channels that gated at low voltages. In contrast, human Cav3.3 isoforms with shorter carboxyl termini were less affected by a prepulse. Therefore, Cav3.3 is similar to high voltage-activated Ca2+ channels in that depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity.