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Presents research using the technique of loading nitr-5 into cells and inducing the release of Ca2+ with a flash of light. Elevation of intracellular Ca2+ concentration directly augmented Ca2+-channel currents in isolated cardiac muscle cells from both frog and guinea pig. Research methods; Statistics.

RGK (Rad-Gem-Rem) GTPases have been described as potent negative regulators of the Ca2+ influx via high-threshold voltage-activated Ca2+ channels. Recent work, mostly performed on Cav1.2 Ca2+ channels, has highlighted the crucial role played by the channel auxiliary Cavβ subunits and identified several GTPase and β-subunit protein domains involved in this regulation. We now extend these conclusions by producing the first complete characterization of the effects of Gem, Rem, and Rem2 on the neuronal Cav2.1 Ca2+ channels expressed with Cavβ1 or Cavβ2 subunits. Current inhibition is limited to a decrease in amplitude with no modification in the voltage dependence or kinetics of the current. We demonstrate that this inhibition can occur for Cavβ constructs with impaired capacity to induce current potentiation, but that it is lost for Cavβ constructs deleted for their β-interaction domain. The RGK C-terminal last ~80 amino acids are sufficient to allow potent current inhibition and in vivo β-subunit/Gem interaction. Interestingly, although Gem and Gem carboxy-terminus induce a completely different pattern of β-subunit cellular localization, they both potently inhibit Cav2.1 channels. These data therefore set the status of neuronal Cav2.1 Ca2+ channel inhibition by RGK GTPases, emphasizing the role of short amino acid sequences of both proteins in β-subunit binding and channel inhibition and revealing a new mechanism for channel inhibition. [ABSTRACT FROM AUTHOR]

Abstract: Calcium influx into cardiac myocytes via voltage-gated Ca channels is a key step in initiating the contractile response. During prolonged depolarizations, toxic Ca2+ overload is prevented by channel inactivation occurring through two different processes identified by their primary trigger: voltage or intracellular Ca2+. In physiological situations, cardiac L-type (CaV1.2) Ca2+ channels inactivate primarily via Ca2+-dependent inactivation (CDI), while neuronal P/Q (CaV2.1) Ca2+ channels use preferentially voltage-dependent inactivation (VDI). In certain situations however, these two types of channels have been shown to be able to inactivate by both processes. From a structural view point, the rearrangement occurring during CDI and VDI is not precisely known, but functional studies have underlined the role played by at least 2 channel sequences: a C-terminal binding site for the Ca2+ sensor calmodulin, essential for CDI, and the loop connecting domains I and II, essential for VDI. The conserved regulation of VDI and CDI by the auxiliary channel subunit strongly suggests that these two mechanisms may use a set of common protein–protein interactions that are influenced by the auxiliary subunit. We will review our current knowledge of these interactions. New data are presented on L-P/Q (CaV1.2/CaV2.1) channel chimera that confirm the role of the I–II loop in VDI and CDI, and reveal some of the essential steps in Ca2+ channel inactivation. [Copyright &y& Elsevier]

Starting from a comparative study of different Ca2+ chelators on the G-protein-induced inhibition of the CaV2.1 Ca channels, we demonstrate that BAPTA and DM-nitrophen are able to interact, in a Ca2+- and lipid-dependent manner, with phospholipid monolayers. Critical insertion pressure and sensitivity to charged lipids indicated that insertion in the lipid film may occur in biological membranes as those found on Xenopus oocytes. This novel property is not found for EGTA and EDTA and may participate to the unusual ability of BAPTA-related molecules to chelate Ca2+ ions in the very close vicinity of the plasma membrane, where most of the Ca2+-dependent signalling triggered by voltage-gated Ca2+ currents occurs. [Copyright &y& Elsevier]

The β subunits of voltage-gated Ca[sup 2+] channels are known to be regulators of the channels' gating properties. Here we report a striking additional function of a β subunit. Screening of chicken cochlear and brain cDNA libraries identified β[sub 4c], a short splice variant of the β[sub 4] subunit. Although β[sub 4c] occurs together with the longer isoforms β[sub 4a] or β[sub 4b] in the brain, eye, heart, and lung, the cochlea expresses exclusively β[sub 4c]. The association of β[sub 4c] with the Ca[sup 2+]-channel α[sub 1] subunit has slight but significant effects on the kinetics of channel activation and inactivation. Yeast two-hybrid and biochemical assays revealed that β[sub 4c] interacts directly with the chromo shadow domain of chromobox protein 2/heterochromatin protein 1γ, (CHCB2/HP1γ), a nuclear protein involved in gene silencing and transcriptional regulation. Coexpression of this protein specifically recruits β[sub 4c] to the nuclei of mammalian cells. Furthermore, β[sub 4c] but not β[sub 4a] dramatically attenuates the genesilencing activity of chromobox protein 2/heterochromatin protein 1γ. The β[sub 4c] subunit is therefore a multifunctional protein that not only constitutes a portion of the Ca[sup 2+] channel but also regulates gene transcription. [ABSTRACT FROM AUTHOR]

Voltage-gated Ca2+ channels are key transducers of cellular excitability and participate in several crucial physiological responses. In vertebrates, 10 Ca2+ channel genes, grouped in 3 families (CaV1, CaV2 and CaV3), have been described and characterized. Insects possess only one member of each family. These genes have been isolated in a limited number of species and very few have been characterized although, in addition to their crucial role, they may represent a collateral target for neurotoxic insecticides. We have isolated the 3 genes coding for the 3 Ca2+ channels expressed in Apis mellifera. This work provides the first detailed characterization of the honeybee T-type CaV3 Ca2+ channel and demonstrates the low toxicity of inhibiting this channel. Comparing Ca2+ currents recorded in bee neurons and myocytes with Ca2+ currents recorded in Xenopus oocytes expressing the honeybee CaV3 gene suggests native expression in bee muscle cells only. High-voltage activated Ca2+ channels could be recorded in the somata of different cultured bee neurons. These functional data were confirmed by in situ hybridization, immunolocalization and in vivo analysis of the effects of a CaV3 inhibitor. The biophysical and pharmacological characterization and the tissue distribution of CaV3 suggest a role in honeybee muscle function. [ABSTRACT FROM AUTHOR]