Xiangyang Wei

The amino terminus of a calcium channel β subunitsets rates of channel inactivation independently of the subunit's effect on activation

There is molecular diversity in both alpha 1 and beta subunits of voltage-gated Ca2+ channels. Coupling between voltage sensing and pore opening of the C-type alpha 1 (alpha 1c) is improved by the type 2 beta subunit (beta 2), and E-type alpha 1 beta complexes inactivate at different rates depending on the nature of beta. We compared the effects of type 1 and 2 beta subunits on activation of the human E-type alpha 1 (alpha 1E) with the effects they have on inactivation, as seen in Xenopus oocytes. The beta subtypes stimulated activation in similar fashion but affected inactivation differently, and even in opposing directions. beta subunits have a common central core but differ in their N- and C-termini and in a central region. N-terminal chimeras between beta 1 and beta 2 subunits that have opposing effects on inactivation resulted in the reciprocal transfer of their effects. We conclude that regulation of activation and inactivation of alpha 1 by beta are separable events and that the N-terminus of beta is one of the structural determinants important in setting the rate and voltage at which an alpha 1 inactivates.

T-type and N-type calcium channels of Xenopus oocytes: evidence for specific interactions with beta subunits.

We used amplifying effects of calcium channel beta subunits to identify endogenous calcium channels in Xenopus oocytes. Expression of rat brain beta 4 increased macroscopic endogenous current magnitude with a small effect on kinetics. In contrast, expression of rat brain/cardiac beta 2 produced a much larger increase in current magnitude and dramatically slowed current decay. Low concentrations of omega-conotoxin GVIA irreversibly blocked currents in both uninjected and beta 2-injected oocytes. Single channel recordings revealed both T- and N-type calcium channels with conductances of 9 and 18 pS, respectively, in uninjected oocytes and in oocytes expressing either beta subunit. Expression of either beta subunit slowed average current decay of T-type single channels. Slowing of T-type current decay by expression of beta 2 was due to reopening of the channels. N-type single channel average current decay showed little change with expression of beta 4, whereas expression of beta 2 slowed average current decay.

Voltage-dependent inactivation in a cardiac-skeletal chimeric calcium channel.

The loci for inactivation in calcium channel proteins are unknown. Mechanisms for inactivation may be distributed across Ca2+ channel subunits and appear to be complex, multiple and interacting. We took advantage of the properties of chimeras, constructed between cardiac (H4) and skeletal muscle (Sk4) calcium channel α 1 subunits to study the molecular mechanism of inactivation in L‐type calcium channels. Sk1H3, a chimeric construct of these two L‐type calcium channels, was expressed in Xenopus oocytes in the absence of auxiliary subunits. Sk1H3 incorporated repeat I from skeletal muscle α 1, and repeats II, III, IV from heart α 1, subunit. Sk1H3 inactivated faster (τ ≈ 300 ms) and more fully than the wild‐type H4 with Ba2+ ions as the charge carrier. Thus, inactivation of Sk1H3 was 90% complete after a 5‐s conditioning pulse at +20 mV while inactivation of H4 was only 37% complete. Sk1H3 inactivation also developed at more negative potentials with E 0.5 = −15 mV as compared to E 0.5 = −5 mV for H4. In the presence of external calcium ions, the extent of inactivation significantly increased from 37 to 83% for H4 while inactivation of Sk1H3 was only slightly increased. Inactivation with Ba2+ as the charge carrier was confirmed at the single‐ channel level where averaged single‐channel ensembles showed a similar rate of inactivation. Collectively, these observations demonstrate that Sk1H3 inactivation appears to have a prominent voltage‐dependent component. Whether Sk1H3 inactivation involves interactions within repeat I alone or interactions between repeat I and site(s) located in the three other repeats of the α 1 subunit has yet to be determined.