Localization Of Cav1 Research Articles

Role Of Calmodulin On The Binding Of Alpha Actinin And C-terminus Of CaV1.2 Ca2+ Channel

Cav1.2 is the most prominent L-type voltage dependent Ca2+ channel in heart and brain constituting ∼80% of total L-type channels. L-type channel controls calcium influx and its regulation by alpha-actinin. The exact function of alpha-actinin in the cell is not clear. However, our studies have shown that alpha-actinin is critical for the activity of L-type Ca2+ channels as it links membrane to the cytoskeleton. We seek to understand the molecular details of how specific channel subunits or associated proteins, such as alpha-actinin and the ubiquitous Ca2+-binding protein calmodulin, regulate the function of voltage-gated Ca2+ channels in brain and heart. In the present study, we employed biochemical and fluorescence anisotropy techniques to identify the exact binding site for alpha-actinin on the cytoplasmic C-terminal tail of the Cav1.2. Our findings indicate that the residues 1507-1733 of alpha1 subunit serves as a common binding region for alpha-actinin and CaM under Ca2+-depleted condition. Intriguingly, this binding is directly antagonized by Ca2+/calmodulin. Furthermore, we investigated the affinity of alpha-actinin for different peptides within 1534-1697 fragments of Cav1.2 and observed affinities in the high nM range with a peptides corresponding to residues 1614-1635 and 1644-1670. We further observed that these two peptides (1614-1635 and 1644-1670) competitively bind to alpha actinin. We have also investigated the affinities of different domains of alpha-actinin for these different peptides (1588-1675 fragments) of the C-terminal tail of the Cav1.2. Thus suggesting that alpha-actinin may play a role in both the localization of Cav1.2 and its modulation by Ca2+. This is a critical negative feed-back mechanism that curbs the influx of calcium, which is a potent signaling molecule inside the cell.

Stable Membrane Expression of Postsynaptic CaV1.2 Calcium Channel Clusters Is Independent of Interactions with AKAP79/150 and PDZ Proteins

In neurons L-type calcium currents contribute to synaptic plasticity and to activity-dependent gene regulation. The subcellular localization of Ca(V)1.2 and its association with upstream and downstream signaling proteins is important for efficient and specific signal transduction. Here we tested the hypothesis that A-kinase anchoring proteins (AKAPs) or PDZ-proteins are responsible for the targeting and anchoring of Ca(V)1.2 in the postsynaptic compartment of glutamatergic neurons. Double-immunofluorescence labeling of hippocampal neurons transfected with external HA epitope-tagged Ca(V)1.2 demonstrated that clusters of membrane-incorporated Ca(V)1.2-HA were colocalized with AKAP79/150 but not with PSD-95 in the spines and shafts of dendrites. To disrupt the interactions with these scaffold proteins, we mutated known binding sequences for AKAP79/150 and PDZ proteins in the C terminus of Ca(V)1.2-HA. Unexpectedly, the distribution pattern, the density, and the fluorescence intensity of clusters were similar for wild-type and mutant Ca(V)1.2-HA, indicating that interactions with AKAP and PDZ proteins are not essential for the correct targeting of Ca(V)1.2. In agreement, brief treatment with NMDA (a chemical LTD paradigm) caused the degradation of PSD-95 and the redistribution of AKAP79/150 and alpha-actinin from dendritic spines into the shaft, without a concurrent loss or redistribution of Ca(V)1.2-HA clusters. Thus, in the postsynaptic compartment of hippocampal neurons Ca(V)1.2 calcium channels form signaling complexes apart from those of glutamate receptors and PSD-95. Their number and distribution in dendritic spines is not altered upon NMDA-induced disruption of the glutamate receptor signaling complex, and targeting and anchoring of Ca(V)1.2 is independent of its interactions with AKAP79/150 and PDZ proteins.

Abstract 5309: beta-Arrestin 1 (bArr1) and Ankyrin 2 (ANK2) Regulate Cav1.2 Trafficking through Complex G Protein Mediated Signaling

Mechanisms governing the trafficking of cardiac voltage dependent calcium channels (Ca v 1.2) remain largely unknown. We have previously shown that neuronal calcium channels are internalized into clathrin coated vesicles upon G protein coupled receptor (GPCR) activation. Here, we hypothesize that GPCR mediated signaling regulate Ca v 1.2 trafficking and surface expression through interaction with cytoskeletal and signaling molecules. Methods : Sequential immunoprecipitation in adult rat cardiomyocytes was used to investigate the interaction between Ca v 1.2, cytoskeletal and signaling molecules. Live cell imaging and immunofluorescence microscopy were used to measure changes in the cellular localization of Ca v 1.2 and these proteins upon activation of GPCR signaling. Results : Cav1.2 interacts with ANK2, βArr1 and spectrin. Sustained β adrenergic receptor (βAR) activation increases Cav1.2/spectrin and decreases Ca v 1.2/ANK2 and Ca v 1.2/βArr1 interactions. Sustained βAR activation induces rapid (<5 min) Cav1.2 internalization and dissociation from ANK2 as Ca v 1.2/ANK2 co-localization is markedly reduced (−58% p<0.01) in isoproterenol treated myocytes. A cell permeant peptide (1.4 μg/ml) that disrupts Ca v 1.2/ANK2 interaction decreases (−40±12%, p<0.01) Cav1.2 cell surface expression. Pretreatment with pertussis toxin prevents Ca v 1.2 internalization suggesting that G i/o mediates this response. Similarly, Src kinase inhibition reduces (by 90%) Ca v 1.2 internalization. Differential labeling of pre-existing Ca v 1.2 at the cell membrane and newly inserted Ca v 1.2 using fluorophore conjugated dihydropyridines reveal that activation of G i/o proteins underlies Ca v 1.2 internalization and insertion. In contrast, activation of the muscarinic M2 receptor (10μM carbachol) causes only insertion of new channels (n=14) and fails to alter the association of Ca v 1.2 with ANK2 and spectrin. Finally PI3 kinase inhibition (10nM wortmannin) prevents Ca v 1.2 membrane insertion. Conclusions : Our findings reveal novel signaling mechanisms that regulate Ca v 1.2 trafficking, internalization and membrane insertion in a receptor subtype dependent manner. These mechanisms may be central to pathologies associated with a hyperadrenergic state. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Neuronal localization of Ca v1.2 L-type calcium channels in the rat basolateral amygdala

L-type high-voltage-activated calcium channels are involved in the conduction and integration of electrical activity and associative long-term potentiation in the basolateral amygdalar complex (BLC). However, little is known about the neuronal localization of these channels in this brain region. We used immunohistochemical techniques to determine which cell types in the BLC express the Ca v1.2 subtype of L-type calcium channels. Immunofluorescence experiments demonstrated that Ca v1.2 calcium channels were mainly found in somata and dendrites of pyramidal neurons that exhibited immunoreactivity for calcium/calmodulin-dependent protein kinase II (CaMK). Only a few parvalbumin-positive and calretinin-positive interneurons exhibited Ca v1.2 immunoreactivity. The presence of high levels of Ca v1.2 immunoreactivity in BLC pyramidal cells is consistent with physiological findings showing that calcium entry through L-type calcium channels in pyramidal cell dendrites in the lateral amygdala is required for associative LTP and the conversion of synaptic events into long-term emotional memory.

Altered localization of Cav1.2 (L‐type) calcium channels in nerve fibers, Schwann cells, odontoblasts, and fibroblasts of tooth pulp after tooth injury

We have determined the localization of Cav1.2 (L-Type) Ca2+ channels in the cells and nerve fibers in molars of normal or injured rats. We observed high levels of immunostaining of L-type Ca2+ channels in odontoblast cell bodies and their processes, in fibroblast cell bodies and in Schwann cells. Many Cav1.2-containing unmyelinated and myelinated axons were also present in root nerves and proximal branches in coronal pulp, but were usually missing from nerve fibers in dentin. Labeling in the larger fibers was present along the axonal membrane, localized in axonal vesicles, and in nodal regions. After focal tooth injury, there is a marked loss of Cav1.2 channels in injured teeth. Immunostaining of Cav1.2 channels was lost selectively in nerve fibers and local cells of the tooth pulp within 10 min of the lesion, without loss of other Cav channel or pulpal labels. By 60 min, Cav1.2 channels in odontoblasts were detected again but at levels below controls, whereas fibroblasts were labeled well above control levels, similar to upregulation of Cav1.2 channels in astrocytes after injury. By 3 days after the injury, Cav1.2 channels were again detected in nerve fibers and immunostaining of fibroblasts and odontoblasts had returned to control levels. These findings provide new insight into the localization of Cav1.2 channels in dental pulp and sensory fibers, and demonstrate unexpected plasticity of channel distribution in response to nerve injury.

Localization of the calcium channel subunits Cav1.2 (alpha1C) and Cav2.3 (alpha1E) in the mouse organ of Corti.

Voltage-activated Ca2+ channels play an important role in synaptic transmission, signal processing and development. The immunohistochemical localization of Cav1.2 (alpha1C) and Cav2.3 (alpha1E) Ca2+ channels was studied in the developing and adult mouse organ of Corti using subunit-specific antibodies and fluorescent secondary antibodies with cochlear cryosections. Cav1.2 immunoreactivity has been detected from postnatal day 14 (P14) onwards at the synapses between cholinergic medial efferents and outer hair cells as revealed by co-staining with anti-synaptophysin and anti-choline acetyltransferase. Most likely the Cav1.2 immunoreactivity was located presynaptically at the site of contact of the efferent bouton with the outer hair cell which suggests a role for class C L-type Ca2+ channels in synaptic transmission of the medial efferent system. The localization of the second Ca2+ channel tested, Cav2.3, showed a pronounced change during cochlear development. From P2 until P10, Cav2.3 immunoreactivity was found in the outer spiral bundle followed by the inner spiral bundle, efferent endings and by medial efferent fibers. Around P14, Cav2.3 immunoreactivity disappeared from these structures and from P19 onwards it was observed in the basal poles of the outer hair cell membranes.