ABSTRACT
L-type voltage gated calcium channels (VGCCs) interact with a variety of proteins that modulate both their function and localization. A-Kinase Anchoring Proteins (AKAPs) facilitate L-type calcium channel phosphorylation through ß adrenergic stimulation. Our previous work indicated a role of neuronal AKAP79/150 in the membrane targeting of Ca(V)1.2 L-type calcium channels, which involved a proline rich domain (PRD) in the intracellular II-III loop of the channel.(1) Here, we show that mutation of proline 857 to alanine (P857A) into the PRD does not disrupt the AKAP79-induced increase in Ca(v)1.2 membrane expression. Furthermore, deletion of two other PRDs into the carboxy terminal domain of Ca(V)1.2 did not alter the targeting role of AKAP79. In contrast, the distal carboxy terminus region of the channel directly interacts with AKAP79. This protein-protein interaction competes with a direct association of the channel II-III linker on the carboxy terminal tail and modulates membrane targeting of Ca(V)1.2. Thus, our results suggest that the effects of AKAP79 occur through relief of an autoinhibitory mechanism mediated by intramolecular interactions of Ca(v)1.2 intracellular regions.
Subject(s)
A Kinase Anchor Proteins/metabolism , Calcium Channels, L-Type/chemistry , Calcium Channels, L-Type/metabolism , Protein Subunits/chemistry , Protein Subunits/metabolism , Amino Acid Sequence , Animals , Calcium Channels, L-Type/genetics , Cell Line, Transformed , Gene Deletion , Humans , Mice , Molecular Sequence Data , Mutation, Missense , Oocytes , Patch-Clamp Techniques , Proline/metabolism , Proline-Rich Protein Domains , Protein Interaction Domains and Motifs , Protein Subunits/genetics , Protein Transport/genetics , XenopusABSTRACT
Inositol 1,4,5-trisphosphate receptors (IP3Rs) release calcium ions, Ca2+, from intracellular stores, but their roles in mediating Ca2+ entry are unclear. IP3 stimulated opening of very few (1.9 +/- 0.2 per cell) Ca2+-permeable channels in whole-cell patch-clamp recording of DT40 chicken or mouse B cells. Activation of the B cell receptor (BCR) in perforated-patch recordings evoked the same response. IP3 failed to stimulate intracellular or plasma membrane (PM) channels in cells lacking IP3R. Expression of IP3R restored both responses. Mutations within the pore affected the conductances of IP3-activated PM and intracellular channels similarly. An impermeant pore mutant abolished BCR-evoked Ca2+ signals, and PM IP3Rs were undetectable. After introduction of an alpha-bungarotoxin binding site near the pore, PM IP3Rs were modulated by extracellular alpha-bungarotoxin. IP(3)Rs are unusual among endoplasmic reticulum proteins in being also functionally expressed at the PM, where very few IP3Rs contribute substantially to the Ca2+ entry evoked by the BCR.
Subject(s)
Calcium Channels/metabolism , Calcium Signaling , Calcium/metabolism , Cell Membrane/metabolism , Ion Channel Gating , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , B-Lymphocytes/metabolism , Bungarotoxins/metabolism , Bungarotoxins/pharmacology , Calcium Channels/genetics , Cells, Cultured , Chickens , Electric Conductivity , Endoplasmic Reticulum/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Inositol 1,4,5-Trisphosphate Receptors , Mice , Nuclear Envelope/metabolism , Patch-Clamp Techniques , Point Mutation , Rats , Receptors, Antigen, B-Cell/metabolism , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/genetics , TransfectionABSTRACT
The inhibition of N-type calcium channels by opioid receptor like receptor 1 (ORL1) is a key mechanism for controlling the transmission of nociceptive signals. We recently reported that signaling complexes consisting of ORL1 receptors and N-type channels mediate a tonic inhibition of calcium entry. Here we show that prolonged ( approximately 30 min) exposure of ORL1 receptors to their agonist nociceptin triggers an internalization of these signaling complexes into vesicular compartments. This effect is dependent on protein kinase C activation, occurs selectively for N-type channels and cannot be observed with mu-opioid or angiotensin receptors. In expression systems and in rat dorsal root ganglion neurons, the nociceptin-mediated internalization of the channels is accompanied by a significant downregulation of calcium entry, which parallels the selective removal of N-type calcium channels from the plasma membrane. This may provide a new means for long-term regulation of calcium entry in the pain pathway.
Subject(s)
Calcium Channels, N-Type/physiology , Pain/physiopathology , Receptors, Opioid/physiology , Aniline Compounds , Animals , Calcium Channels, N-Type/genetics , Cells, Cultured , Down-Regulation/drug effects , Down-Regulation/physiology , Electrophysiology , Fluorescent Dyes , Ganglia, Spinal/cytology , Ganglia, Spinal/metabolism , Ganglia, Spinal/physiology , Image Processing, Computer-Assisted , Immunohistochemistry , Mice , Mice, Knockout , Microscopy, Confocal , Receptors, Opioid/agonists , Receptors, Opioid/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Xanthenes , Nociceptin ReceptorABSTRACT
It has been suggested that the auxiliary subunits of high voltage-activated (HVA) calcium channels modulate T-type, low voltage-activated (LVA) calcium channels. Such a regulation has yet to be documented, especially because there has been no biochemical characterization of T-channels. To monitor total protein levels and plasma membrane expression of T-channels in living cells, external epitopes (hemagglutinin, FLAG) were introduced into human recombinant Ca(V)3 channels that were also N-terminally fused to green fluorescent protein. Utilizing Western blot techniques, fluorescence flow cytometry, immunofluorescence, luminometry, and electrophysiology, we describe here that beta(1b) and alpha(2)-delta(1) subunits enhance the level of Ca(V)3 proteins as well as their plasma membrane expression in various expression systems. We also report that, in both Xenopus oocytes and mammalian cells, the alpha(2)-delta(1) subunits increase by at least and beta(1b) 2-fold the current density of Ca(V)3 channels with no change in the electrophysiological properties. Altogether, these data indicate that HVA auxiliary subunits modulate Ca(V)3 channel surface expression, suggesting that the membrane targeting of HVA and LVA alpha(1) subunits is regulated dynamically through the expression of a common set of regulatory subunits.
Subject(s)
Calcium Channels, T-Type/metabolism , Cell Membrane/metabolism , Protein Subunits/metabolism , Animals , Calcium Channels, T-Type/chemistry , Calcium Channels, T-Type/genetics , Cell Line , Cricetinae , Electrophysiology , Epitopes , Humans , Membrane Potentials/physiology , Oocytes/physiology , Protein Structure, Secondary , Protein Subunits/chemistry , Protein Subunits/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , XenopusABSTRACT
Accurate calcium signaling requires spatial and temporal coordination of voltage-gated calcium channels (VGCCs) and a variety of signal transduction proteins. Accordingly, regulation of L-type VGCCs involves the assembly of complexes that include the channel subunits, protein kinase A (PKA), protein kinase A anchoring proteins (AKAPs), and beta2-adrenergic receptors, although the molecular details underlying these interactions remain enigmatic. We show here, by combining extracellular epitope splicing into the channel pore-forming subunit and immunoassays with whole cell and single channel electrophysiological recordings, that AKAP79 directly regulates cell surface expression of L-type calcium channels independently of PKA. This regulation involves a short polyproline sequence contained specifically within the II-III cytoplasmic loop of the channel. Thus we propose a novel mechanism whereby AKAP79 and L-type VGCCs function as components of a biosynthetic mechanism that favors membrane incorporation of organized molecular complexes in a manner that is independent of PKA phosphorylation events.
