Kinetic modification of the α1I subunit-mediated T-type Ca2+ channel by a human neuronal Ca2+ channel γ subunit

1. Voltage-sensitive Ca2+ channels (VSCCs) are often heteromultimeric complexes. The VSCC subtype specifically expressed by skeletal muscle has long been known to contain a gamma subunit, gamma (1), that is only expressed in this tissue. Recent work, initiated blv the identification of the mutation present in the stargazer mouse, has led to the identification of a series of novel potential Ca2+ channel gamma subunits expressed in the CNS. 2. Based on bioinformatic techniques we identified and cloned the human gamma (2), gamma (3) and gamma (4) subunits. 3. TayMan analysis was used to quantitatively characterise the mRNA expression patter ns of all the gamma subunits. All three subunits were extensively expressed in adult brain with overlapping but subunit-specific distributions, gamma (2) and gamma (3) were almost entirely restricted to the brain, but gamma (4) expression was seen in a broad range of peripheral tissues 4. Using a myc epitope the gamma (2) subunit was tagged both intracellularly at the C-terminus: and on a predicted extracellular site between the first and second transmembrane domains. The cellular distribution was then examined immunocytochemically, which indicated that a substantial proportion of the cellular pool of the gamma (2) subunit was present on the plasma membrane and provided initial evidence for the predicted transmembrane topology of the gamma subunits. 5. Using co-transfection techniques we investigated the functional effects of each of the gamma subunits on the biophysics of the T-type VSCC encoded by the alpha (1I) subunit. This revealed a substantially slowed rate of deactivation in the presence of gamma (2). In contrast, there significant corresponding effect of either gamma (3) or gamma (4) on alpha (1I) subunit-mediated currents.