Molecular Characterization of Two Members of the T‐Type Calcium Channel Family

Abstract

In this chapter we review our recent studies on the cloning of two novel cDNAs (α1G and α1H), and present electrophysiological evidence that they encode low voltage‐activated, T‐type calcium channels (Ca_vT.1 and Ca_vT.2, respectively). The nucleotide sequences of these T channels are very different from high voltage‐activated Ca²⁺ channels, which explains why they were not cloned earlier using homology‐based strategies. We used a bioinformatic approach, cloning the first fragment in silico. We then used this fragment to screen human heart and rat brain λgt10 libraries, leading to the cloning of two full‐length cDNAs derived from distinct genes (CACNA1G and CACNA1H). The deduced amino acid sequences of the T channels (α1G and α1H) are also very different from previously cloned Ca²⁺ and Na⁺ channels; however, there are regions of structural similarity. For example, the T channels also contain four repeats, and within each repeat there are six putative membrane‐spanning regions and a pore loop. Expression of these cloned channels in either Xenopus oocytes or HEK‐293 cells leads to the formation of typical T‐type currents. As observed for native T currents, these channels activate at potentials near the resting membrane potential, inactivate rapidly, deactivate slowly, and have a tiny single‐channel conductance. The currents generated by α1G and α1H are nearly identical in terms of their voltage dependence and kinetics. We present preliminary evidence that nickel may serve as a valuable tool in discriminating between these subtypes.