VOLTAGE-DEPENDENT INACTIVATION IN A CARDIAC-SKELETAL CHIMERIC CALCIUM-CHANNEL

The loci for inactivation in calcium channel proteins are unknown. Mec hanisms for inactivation may be distributed across Ca2+ channel subuni ts and appear to be complex, multiple and interacting, We took advanta ge of the properties of chimeras, constructed between cardiac (H4) and skeletal muscle (Sk4) calcium channel alpha(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 expres sed in Xenopus oocytes in the absence of auxiliary submits. Sk1H3 inco rporated repeat I from skeletal muscle alpha(1) and repeats II, III, I V from heart alpha(1) subunit. Sk1H3 inactivated faster (tau approxima te to = 300 ms) and more fully than the wild-type H4 with Ba2+ ions as the charge carrier. Thus, inactivation of Sk1H3 was 90% complete afte r a 5-s conditioning pulse at +20 mV while inactivation of H4 was only 37% complete. Sk1H3 inactivation also developed at more negative pote ntials with E(0.5) = -15 mV as compared to E(0.5) = -5 mV for H4. In t he presence of external calcium ions, the extent of inactivation signi ficantly increased from 37 to 83% for H4 while inactivation of Sk1H3 w as only slightly increased. Inactivation with Ba2+ as the charge carri er was confirmed at the single- channel level where averaged single-ch annel ensembles showed a similar rate of inactivation. Collectively, t hese observations demonstrate that Sk1H3 inactivation appears to have a prominent voltage-dependent component. Whether Sk1H3 inactivation in volves interactions within repeat I alone or interactions between repe at I and site(s) located in the three other repeats of the alpha(1) su bunit has yet to be determined.