Auxiliary subunit regulation of high-voltage activated calcium channels expressed in mammalian cells

The effects of auxiliary calcium channel subunits on the expression and functional properties of high-voltage activated (HVA) calcium channels have been studied extensively in the Xenopus oocyte expression system, but are less completely characterized in a mammalian cellular environment. Here, we provide the first systematic analysis of the effects of calcium channel beta and alpha(2)-delta subunits on expression levels and biophysical properties of three different types (Ca(v)1.2, Ca(v)2.1 and Ca(v)2.3) of HVA calcium channels expressed in tsA-201 cells. Our data show that Ca(v)1.2 and Ca(v)2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel beta subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel alpha(1) subunit, and beta(3) subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the alpha(2)-delta subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Ca(v)2 channel family, appears to act synergistically with beta subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by beta and by alpha(2)-delta subunits. However, whereas pronounced regulation of inactivation characteristics was seen with the majority of the auxiliary subunits, effects on voltage dependence of activation were only small (< 5 mV). Overall, through a systematic approach, we have elucidated a previously underestimated role of the alpha(2)-delta(1) subunit with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes

Regulation of cardiac L-type Ca2+ channel by coexpression of G\u3b1s in Xenopus oocytes

Activation of G(alpha s) via beta-adrenergic receptors enhances the activity of cardiac voltage-dependent Ca2+ channels of the L-type, mainly via protein kinase A (PKA)-dependent phosphorylation. Contribution of a PKA-independent effect of G(alpha s) has been proposed but remains controversial. We demonstrate that, in Xenopus oocytes, antisense knockdown of endogenous G(alpha s) reduced, whereas coexpression of G(alpha s) enhanced, currents via expressed cardiac L-type channels, independently of the presence of the auxiliary subunits alpha2/delta or beta2A. Coexpression of G(alpha s) did not increase the amount of alpha1C protein in whole oocytes or in the plasma membrane (measured immunochemically). Activation of coexpressed beta2 adrenergic receptors did not cause a detectable enhancement of channel activity; rather, a small cAMP-dependent decrease was observed. We conclude that coexpression of G(alpha s), but not its acute activation via beta-adrenergic receptors, enhances the activity of the cardiac L-type Ca2+ channel via a PKA-independent effect on the alpha1C subunit

The Calcium Store Sensor, STIM1, Reciprocally Controls Orai and CaV1.2 Channels

Calcium signals, pivotal in controlling cell function, can be generated by calcium entry channels activated by plasma membrane depolarization or depletion of internal calcium stores. We reveal a regulatory link between these two channel subtypes mediated by the ubiquitous calcium-sensing STIM proteins. STIM1 activation by store depletion or mutational modification strongly suppresses voltage-operated calcium (Ca(V)1.2) channels while activating store-operated Orai channels. Both actions are mediated by the short STIM-Orai activating region (SOAR) of STIM1. STIM1 interacts with Ca(V)1.2 channels and localizes within discrete endoplasmic reticulum/plasma membrane junctions containing both Ca(V)1.2 and Orai1 channels. Hence, STIM1 interacts with and reciprocally controls two major calcium channels hitherto thought to operate independently. Such coordinated control of the widely expressed Ca(V)1.2 and Orai channels has major implications for Ca(2+) signal generation in excitable and nonexcitable cells

Direct measurement of single-channel Ca2+ currents in bullfrog hair cells reveals two distinct channel subtypes

To confer their acute sensitivity to mechanical stimuli, hair cells employ Ca2+ ions to mediate sharp electrical tuning and neurotransmitter release. We examined the diversity and properties of voltage-gated Ca2+ channels in bullfrog saccular hair cells by means of perforated and cell-attached patch-clamp techniques. Whole-cell Ca2+ current records provided hints that hair cells express L-type as well as dihydropyridine-insensitive Ca2+ currents.Single Ca2+ channel records confirmed the presence of L-type channels, and a distinct Ca2+ channel, which has sensitivity towards ω-conotoxin GVIA. Despite its sensitivity towards ω-conotoxin GVIA, the non-L-type channel cannot necessarily be considered as an N-type channel because of its distinct voltage-dependent gating properties.Using 65 mm Ca2+ as the charge carrier, the L-type channels were recruited at about –40 mV and showed a single-channel conductance of 13 pS. Under similar recording conditions, the non-L-type channels were activated at ∼–60 mV and had a single-channel conductance of ∼16 pS.The non-L-type channel exhibited at least two fast open time constants (τo = 0.2 and 5 ms). In contrast, the L-type channels showed long openings (τo =∼23 ms) that were enhanced by Bay K 8644, in addition to the brief openings (τo = 0.3 and 10 ms).The number of functional channels observed in patches of similar sizes suggests that Ca2+ channels are expressed singly, in low-density clusters (2–15 channels) and in high-density clusters (20–80 channels). Co-localization of the two channel subtypes was observed in patches containing low-density clusters, but was rare in patches containing high-density clusters.Finally, we confirmed the existence of two distinct Ca2+ channel subtypes by using immunoblot and immunohistochemical techniques