Abstract
We have investigated the molecular determinants that mediate the differences in voltage-dependent inactivation properties between rapidly inactivating (R-type) alpha(1E) and noninactivating (L-type) alpha(1C) calcium channels. When coexpressed in human embryonic kidney cells with ancillary beta(1b) and alpha(2)-delta subunits, the wild type channels exhibit dramatically different inactivation properties; the half-inactivation potential of alpha(1E) is 45 mV more negative than that observed with alpha(1C), and during a 150-ms test depolarization, alpha(1E) undergoes 65% inactivation compared with only about 15% for alpha(1C). To define the structural determinants that govern these intrinsic differences, we have created a series of chimeric calcium channel alpha(1) subunits that combine the major structural domains of the two wild type channels, and we investigated their voltage-dependent inactivation properties. Each of the four transmembrane domains significantly affected the half-inactivation potential, with domains II and III being most critical. In particular, substitution of alpha(1C) sequence in domains II or III with that of alpha(1E) resulted in 25-mV negative shifts in half-inactivation potential. Similarly, the differences in inactivation rate were predominantly governed by transmembrane domains II and III and to some extent by domain IV. Thus, voltage-dependent inactivation of alpha(1E) channels is a complex process that involves multiple structural domains and possibly a global conformational change in the channel protein.
Highlights
The influx of calcium through neuronal voltage-gated calcium channels regulates a wide range of cellular processes, including neurotransmitter release, activation of Ca2ϩ-dependent enzymes and second messenger cascades, gene regulation, and proliferation
A study by Zhang et al [29] has revealed that the domain I S6 region is a critical determinant of the differences in voltage-dependent inactivation properties observed with marine ray ␣1E and rabbit brain ␣1A calcium channels
In order to more systematically examine the molecular determinants governing calcium channel inactivation, we have created a series of chimerical calcium channel ␣1 subunits, which combine the structural features of rapidly inactivating ␣1E and noninactivating ␣1C calcium channels, expressed them transiently in Human embryonic kidney (HEK) cells, and assessed their inactivation properties via patch clamp
Summary
The influx of calcium through neuronal voltage-gated calcium channels regulates a wide range of cellular processes, including neurotransmitter release, activation of Ca2ϩ-dependent enzymes and second messenger cascades, gene regulation, and proliferation. Several individual amino acid substitutions throughout the calcium channel ␣1 subunit, including the domain I-II linker region, the proximal carboxyl-terminal region, and the S6 regions in domains III and IV (7, 11, 30 –36) have been shown to reduce or abolish voltage-dependent inactivation. These observations suggest that voltage-dependent inactivation of calcium channels may perhaps involve multiple structural elements. We hypothesize that the molecular mechanisms underlying fast voltage-dependent inactivation in neuronal calcium channels may be analogous to the slower C-type inactivation process common to many types of potassium channels [23, 37, 38]
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