The Cavβ subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels

Abstract

7 3 a r t I C l e SThe level of expression of voltage-gated calcium channels at the plasma membrane is a key regulator of calcium homeostasis in excitable cells, and of downstream effects such as calcium-dependent transcription 1,2 . Members of the high voltage-activated (HVA) calcium channel family are heteromultimeric protein complexes that contain a pore-forming α 1 subunit that defines the channel subtype, plus ancillary α 2 -δ and β subunits that not only alter the function of the α 1 subunit but also regulate the trafficking of the channel complex to the plasma membrane 3-8 . The mammalian genome encodes four different types of Cavβ subunit that have distinct spatial and temporal expression patterns [4][5][6] . With the exception of Cavβ 2a , these subunits are cytoplasmic proteins that physically bind to a region in the α 1 subunit domain I-II linker that is highly conserved among all HVA calcium channels and is termed the alpha interaction domain (AID) 7 . Crystal structure data show that the Cavβ subunit contains interacting SH3 and guanylate kinase domains, with the latter participating in high-affinity binding to the AID region [8][9][10] . The physiological consequences of this interaction are underscored by gene knockout studies showing that deletion of the Cavβ 1a or Cavβ 2a subunits causes embryonic lethality 11,12 and by findings that a premature stop mutation in Cavβ 4 causes an epileptic phenotype in mice 13 .It has been suggested that the Cavβ subunit masks an endoplasmic reticulum retention signal on the Cav2.1 α 1 subunit 14 , thereby leading to increased cell surface expression of P/Q-type channels. However, no specific endoplasmic reticulum retention motif in the AID and surrounding regions of the α 1 subunit has been identified, and removing the AID motif in the I-II linker of Cav2.1 does not increase current amplitude in the absence of Cavβ (ref. 15). Moreover, it is not clear whether different HVA calcium channel isoforms share common retention motifs. Here we show that Cav1.2 (L-type) calcium channels contain an endoplasmic reticulum retention motif in the proximal C-terminal region, and we provide evidence that the Cavβ subunit has a role in regulating proteasomal degradation of these channels. Our data show that the Cavβ subunit acts as a molecular switch that prevents the ubiquitination of the channels and their targeting to the ERAD complex and thereby regulates their expression at the plasma membrane. RESULTS Cavb regulates endoplasmic reticulum retention of Cav1.2We first performed an ELISA assay involving a Cav1.2 channel construct tagged with an extracellular hemagglutinin (HA) epitope (Fig. 1a). We compared immunoluminescence between permeabilized and nonpermeabilized cells, which allowed us to quantify the relative proportion of Cav1.2 channels that were inserted into the plasma membrane. Coexpression with the Cavβ 1b or Cavβ 2a subunit mediated a significant increase in the fraction of Cav1.2 channels at the cell surface (Fig. 1a and data not shown). This was confirmed by HA...

14-3-3τ Promotes Surface Expression of Cav2.2 (α1B) Ca2+ Channels

“…In particular, the Cav ␤ subunit masks an unidentified endoplasmic reticulum (ER) retention signal on the ␣1 subunit and thus increases the trafficking of Cav channels to the plasma membrane (7,8). Moreover, the ␤ subunits may promote the Cav1 and Cav2.2 channel expression by preventing their degradation via the proteasomal pathway (9,10).…”

Targeting the Transient Receptor Potential Vanilloid Type 1 (TRPV1) Assembly Domain Attenuates Inflammation-induced Hypersensitivity

“…TRPV1-YFP deletion mutants were made by loopout mutagenesis using partially overlapping primers (15) to delete residues 716 -733, 734 -751, or 752-772. The CD4-AA cell surface marker was a generous gift from Dr. Zamponi and is described in Altier et al (16). The CD4-Nt2 was made by subcloning the coding sequence corresponding to residues 155-308 of rat TRPV1into CD4-AA; the CD4-Ct was made by subcloning the full-length C terminus (682-838) into CD4-AA using NotI and XbaI sites.…”

“…Cell Culture and Transfection-Human embryonic kidney tsA-201 cells were cultured as described previously (16). Cells were transfected at ϳ50% confluence using the calcium phosphate method.…”

“…Electrophysiology-Whole-cell patch clamp experiments on HEK cells were performed 16 -24 h after transfection with calcium phosphate (16). The internal pipette solution contained 140 mM CsCl, 5 mM NaCl, 1 mM MgCl 2 , 1 mM BAPTA, 0.45 mM CaCl 2 and was adjusted to pH 7.2 with CsOH.…”

“…As a control, the effect of CD4-AA, the membrane-tethered construct not fused to the TRPV1 C terminus was compared with CD4-Ct (16).…”