Cloning and expression of the human T-type channel Ca(v)3.3: insights into prepulse facilitation.
The full-length human Ca(v)3.3 (alpha(1I)) 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 5 mM Ca(2+) as charge carrier. From a holding potential of -100 mV, step depolarizations elicited inward currents with an apparent threshold of -70 mV, a peak of -30 mV, and reversed at +40 mV. The kinetics of channel activation, inactivation, deactivation, and recovery from inactivation were very similar to those reported previously for rat Ca(v)3.3. Similar voltage-dependent gating and kinetics were found for truncated versions of human Ca(v)3.3, 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 Ca(v)3.3 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 (+100 mV, 200 ms) increased the fraction of channels that gated at low voltages. In contrast, human Ca(v)3.3 isoforms with shorter carboxyl termini were less affected by a prepulse. Therefore, Ca(v)3.3 is similar to high voltage-activated Ca(2+) channels in that depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity.
Gomora
Juan Carlos
JC
Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.
Murbartián
Janet
J
Arias
Juan Manuel
JM
Lee
Jung-Ha
JH
Perez-Reyes
Edward
E
D000595:Amino Acid Sequence; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D001921:Brain / Q000378:metabolism; D020747:Calcium Channels, T-Type / Q000737:chemistry* / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D013997:Time Factors; D014162:Transfection
C469757:CACNA1I protein, human; D020747:Calcium Channels, T-Type; D018076:DNA, Complementary; D026901:Membrane Transport Proteins