Dataset Information

Dynamic interactions between L-type voltage-sensitive calcium channel Cav1.2 subunits and ahnak in osteoblastic cells.

ABSTRACT: Voltage-sensitive Ca(2+) channels (VSCCs) mediate Ca(2+) permeability in osteoblasts. Association between VSCC alpha(1)- and beta-subunits targets channel complexes to the plasma membrane and modulates function. In mechanosensitive tissues, a 700-kDa ahnak protein anchors VSCCs to the actin cytoskeleton via the beta(2)-subunit of the L-type Ca(v)1.2 (alpha(1C)) VSCC complex. Ca(v)1.2 is the major alpha(1)-subunit in osteoblasts, but the cytoskeletal complex and subunit composition are unknown. Among the four beta-subtypes, the beta(2)-subunit and, to a lesser extent, the beta(3)-subunit coimmunoprecipitated with the Ca(v)1.2 subunit in MC3T3-E1 preosteoblasts. Fluorescence resonance energy transfer revealed a complex between Ca(v)1.2 and beta(2)-subunits and demonstrated their association in the plasma membrane and secretory pathway. Western blot and immunohistochemistry showed ahnak association with the channel complex in the plasma membrane via the beta(2)-subunit. Cytochalasin D exposure disrupted the actin cytoskeleton but did not disassemble or disrupt the function of the complex of L-type VSCC Ca(v)1.2 and beta(2)-subunits and ahnak. Similarly, small interfering RNA knockdown of ahnak did not disrupt the actin cytoskeleton but significantly impaired Ca(2+) influx. Collectively, we showed that Ca(v)1.2 and beta(2)-subunits and ahnak form a stable complex in osteoblastic cells that permits Ca(2+) signaling independently of association with the actin cytoskeleton.

SUBMITTER: Shao Y

PROVIDER: S-EPMC2681378 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Dynamic interactions between L-type voltage-sensitive calcium channel Cav1.2 subunits and ahnak in osteoblastic cells.

Shao Ying Y Czymmek Kirk J KJ Jones Patricia A PA Fomin Victor P VP Akanbi Kamil K Duncan Randall L RL Farach-Carson Mary C MC

American journal of physiology. Cell physiology 20090304 5

Voltage-sensitive Ca(2+) channels (VSCCs) mediate Ca(2+) permeability in osteoblasts. Association between VSCC alpha(1)- and beta-subunits targets channel complexes to the plasma membrane and modulates function. In mechanosensitive tissues, a 700-kDa ahnak protein anchors VSCCs to the actin cytoskeleton via the beta(2)-subunit of the L-type Ca(v)1.2 (alpha(1C)) VSCC complex. Ca(v)1.2 is the major alpha(1)-subunit in osteoblasts, but the cytoskeletal complex and subunit composition are unknown. A ...[more]

PMID: 19261907

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Project description:Key pointsCav1.2 channels maintain activity through interactions with calmodulin (CaM). In this study, activities of the Cav1.2 channel (α1C) and of mutant-derivatives, C-terminal deleted (α1CΔ) and α1CΔ linked with CaM (α1CΔCaM), were compared in the inside-out mode. α1CΔ with CaM, but not without CaM, and α1CΔCaM were active, suggesting that CaM induced channel activity through a dynamic interaction with the channel, even without the distal C-tail. ATP induced α1C activity with CaM and enhanced activity of the mutant channels. Okadaic acid mimicked the effect of ATP on the wildtype but not mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels through their dynamic interactions. ATP effects involve mechanisms both related and unrelated to channel phosphorylation. CaM-linked channels are useful tools for investigating Cav1.2 channels in the inside-out mode; the fast run-down is prevented by only ATP and the slow run-down is nearly absent.AbstractCalmodulin (CaM) plays a critical role in regulation of Cav1.2 Ca2+ channels. CaM binds to the channel directly, maintaining channel activity and regulating it in a Ca2+ -dependent manner. To explore the molecular mechanisms involved, we compared the activity of the wildtype channel (α1C) and mutant derivatives, C-terminal deleted (α1C∆) and α1C∆ linked to CaM (α1C∆CaM). These were co-expressed with β2a and α2δ subunits in HEK293 cells. In the inside-out mode, α1C and α1C∆ showed minimal open-probabilities in a basic internal solution (run-down), whereas α1C∆ with CaM and α1C∆CaM maintained detectable channel activity, confirming that CaM was necessary, but not sufficient, for channel activity. Previously, we reported that ATP was required to maintain channel activity of α1C. Unlike α1C, the mutant channels did not require ATP for activation in the early phase (3-5 min). However, α1C∆ with CaM + ATP and α1C∆CaM with ATP maintained activity, even in the late phase (after 7-9 min). These results suggested that CaM and ATP interacted dynamically with the proximal C-terminal tail of the channel and, thereby, produced channel activity. In addition, okadaic acid, a protein phosphatase inhibitor, could substitute for the effects of ATP on α1C but not on the mutant channels. These results supported the hypothesis that CaM and ATP maintain activity of Cav1.2 channels, further indicating that ATP has dual effects. One maintains phosphorylation of the channel and the other becomes apparent when the distal carboxyl-terminal tail is removed.

Project description:As with other elements of the peripheral auditory system, spiral ganglion neurons display specializations that vary as a function of location along the tonotopic axis. Previous work has shown that voltage-gated K(+) channels and synaptic proteins show graded changes in their density that confers rapid responsiveness to neurons in the high frequency, basal region of the cochlea and slower, more maintained responsiveness to neurons in the low frequency, apical region of the cochlea. In order to understand how voltage-gated calcium channels (VGCCs) may contribute to these diverse phenotypes, we identified the VGCC α-subunits expressed in the ganglion, investigated aspects of Ca(2+)-dependent neuronal firing patterns, and mapped the intracellular and intercellular distributions of seven VGCC α-subunits in the spiral ganglion in vitro. Initial experiments with qRT-PCR showed that eight of the ten known VGCC α-subunits were expressed in the ganglion and electrophysiological analysis revealed firing patterns that were consistent with the presence of both LVA and HVA Ca(2+) channels. Moreover, we were able to study seven of the α-subunits with immunocytochemistry, and we found that all were present in spiral ganglion neurons, three of which were neuron-specific (Ca(V)1.3, Ca(V)2.2, and Ca(V)3.3). Further characterization of neuron-specific α-subunits showed that Ca(V)1.3 and Ca(V)3.3 were tonotopically-distributed, whereas Ca(V)2.2 was uniformly distributed in apical and basal neurons. Multiple VGCC α-subunits were also immunolocalized to Schwann cells, having distinct intracellular localizations, and, significantly, appearing to distinguish putative compact (Ca(V)2.3, Ca(V)3.1) from loose (Ca(V)1.2) myelin. Electrophysiological evaluation of spiral ganglion neurons in the presence of TEA revealed Ca(2+) plateau potentials with slopes that varied proportionately with the cochlear region from which neurons were isolated. Because afterhyperpolarizations were minimal or absent under these conditions, we hypothesize that differential density and/or kinetics of one or more of the VGCC α-subunits could account for observed tonotopic differences. These experiments have set the stage for defining the clear multiplicity of functional control in neurons and Schwann cells of the spiral ganglion.

Dataset Information

Dynamic interactions between L-type voltage-sensitive calcium channel Cav1.2 subunits and ahnak in osteoblastic cells.

Publications

Dynamic interactions between L-type voltage-sensitive calcium channel Cav1.2 subunits and ahnak in osteoblastic cells.

Similar Datasets

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