The Role of Ca2+ Channel Subunit Composition in G Protein-Mediated Inhibition of Ca2+ Channels: a Disstertation
Graduate School of Biomedical Sciences, Department of Pharmacology
Calcium Channels; GTP-Binding Proteins; Signal Transduction; Academic Dissertations
Modulation of Ca2+ channels is an important mechanism for regulation of synaptic strength. However, it is clear that some Ca2+ current types are insensitive to inhibitory modulation mediated by heterotrimeric G proteins (G protein inhibition), and among currents which are sensitive to G protein inhibition, there is great variation in the magnitude of Ca2+ current inhibition between cells of different origin. For the experiments in this dissertation, I utilized recently cloned Ca2+ channels to determine the minimal combination of Ca2+ channel subunits which would confer G protein sensitivity to the recombinant channels. In addition, I examined the role Ca2+ channel auxiliary subunits play in regulation of Ca2+ channel sensitivity to inhibitory G proteins, and whether channels which were sensitive to G protein inhibition were regulated equivalently by the auxiliary subunits. Finally, I investigated possible mechanisms by which these auxiliary subunits modulate G protein-mediated inhibition of Ca2+ channels.
I found that α1A and α1B Ca2+ currents, when expressed in Xenopus oocytes, were sensitive to modulation by G proteins in the absence of any Ca2+ channel auxiliary subunits, while α1C currents were not modulated under the same conditions. I conclude from this data that Ca2+ channel α1 subunits are differentially sensitive to G protein modulation, and the α1 subunit of the class A and B Ca2+ channels is sufficient for G protein modulation.
I also tested the ability of Ca2+ channel auxiliary subunits to modulate the magnitude of G protein-mediated inhibition Ca2+ currents. I found that the Ca2+ channel α2 subunit had no effect on the magnitude of G protein inhibition of α1A and α1B currents. However, the Ca2+ channel β3 subunit eliminated tonic G protein inhibition and sharply reduced the magnitude of muscarinic M2 receptor induced G protein inhibition of both α1A and α1B currents. I found, however, that while the magnitude of α1A and α1B current inhibition was equivalent in the absence of auxiliary subunits, the magnitude of inhibition was greater for the α1B channel after co-expression of the Ca2+ channel β3 subunit. These results indicate that the Ca2+ channel β3 subunit reduces the sensitivity of α1A and α1B Ca2+ channels to voltage-dependent G protein modulation, and does so to a greater extent for α1A channels when compared to α1B Ca2+ channels.
I found that M2 receptor induced inhibition of α1B currents was more voltage-dependent after expression of the Ca2+ channel β3 subunit. Additionally, the rate relief of G protein inhibition dramatically increased after co-expression of the Ca2+ channel β3 subunit. I also co-expressed G protein subunits, and determined that inhibition of both α1B and α1Bβ3 currents was mediated by the G protein βγ subunit. Furthermore, the rate of voltage-dependent relief of G protein βγ subunit induced inhibition increased after co-expression of the Ca2+ channel β3 subunit, similar to the increased rate of relief of the M2 receptor induced G protein inhibition. These data, along with data which demonstrates that G protein inhibition results from the binding of the G protein βγ subunit to the Ca2+ channel (De Waard et al., 1997), indicate that the Ca2+ channel β3 subunit subunit reduces the magnitude of G protein inhibition of α1B Ca2+ currents by increasing the rate of dissociation of the G protein βγ subunit, such that moderate depolarizations used to activate the channel also relieve a large portion of the G protein inhibition.
Roche, JP. The Role of Ca2+ Channel Subunit Composition in G Protein-Mediated Inhibition of Ca2+ Channels: a Disstertation. (1997). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 281. https://escholarship.umassmed.edu/gsbs_diss/281
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