Abstract
Ca(V)beta subunits modulate cell surface expression and voltage-dependent gating of high voltage-activated (HVA) Ca(V)1 and Ca(V)2 alpha1 subunits. High affinity Ca(V)beta binding onto the so-called alpha interaction domain of the I-II linker of the Ca(V)alpha1 subunit is required for Ca(V)beta modulation of HVA channel gating. It has been suggested, however, that Ca(V)beta-mediated plasma membrane targeting could be uncoupled from Ca(V)beta-mediated modulation of channel gating. In addition to Ca(V)beta, Ca(V)alpha2delta and calmodulin have been proposed to play important roles in HVA channel targeting. Indeed we show that co-expression of Ca(V)alpha2delta caused a 5-fold stimulation of the whole cell currents measured with Ca(V)1.2 and Ca(V)beta3. To gauge the synergetic role of auxiliary subunits in the steady-state plasma membrane expression of Ca(V)1.2, extracellularly tagged Ca(V)1.2 proteins were quantified using fluorescence-activated cell sorting analysis. Co-expression of Ca(V)1.2 with either Ca(V)alpha2delta, calmodulin wild type, or apocalmodulin (alone or in combination) failed to promote the detection of fluorescently labeled Ca(V)1.2 subunits. In contrast, co-expression with Ca(V)beta3 stimulated plasma membrane expression of Ca(V)1.2 by a 10-fold factor. Mutations within the alpha interaction domain of Ca(V)1.2 or within the nucleotide kinase domain of Ca(V)beta3 disrupted the Ca(V)beta3-induced plasma membrane targeting of Ca(V)1.2. Altogether, these data support a model where high affinity binding of Ca(V)beta to the I-II linker of Ca(V)alpha1 largely accounts for Ca(V)beta-induced plasma membrane targeting of Ca(V)1.2.
Highlights
JULY 23, 2010 VOLUME 285 NUMBER 30 forming CaV␣1 subunit, CaV1 and CaV2 channels arise from the multimerization of three other proteins [7]: a cytoplasmic CaV subunit, a mostly extracellular CaV␣2␦ subunit, and calmodulin constitutively bound to the C terminus of CaV␣1 (8 –12)
In CaV2.2, the I-II linker is presumed to play a role in this process [17, 18], and mutations within the AID motif eliminated its cell surface expression and biophysical modulation by CaV1b and CaV3 [32]
Co-expression of CaV␣2␦ promoted the trafficking of the CaV␣1 subunit of CaV2.2 in COS-7 cells [45], suggesting that CaV␣2␦ could promote targeting of all high voltage-activated (HVA) CaV␣1 subotide kinase; HA, hemagglutinin; Fluorescence-activated Cell Sorting (FACS), fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate
Summary
Recombinant DNA Techniques—The rabbit CaV1.2 (GenBankTM accession number X15539), the rat CaV3 (GenBankTM accession number M88751) [52], the rat brain CaV␣2b␦ (GenBankTM accession number NM_000722) [53], and the human CaM (GenBankTM accession number M27319) were used. Protein expression of the auxiliary subunits in the stable and transient cell lines were confirmed routinely by Western blotting (see Fig. 2B). CaV1.2 channel in the stable CaV3 stable cell line as compared with a current density of Ϫ41 Ϯ 9 pA/pF (n ϭ 7) for the wild type CaV1.2 channel measured in the same cell line after transient transfection with. Whole cell current traces recorded after the transient expression of the CaV1.2-HA channel and the CaV␣2b␦ in the stable CaV3 cell line. Transient co-expression of Statistical Analysis—Statistical analyses were performed the HA-tagged CaV1.2 subunit in the stable CaV3 cell line using the built-in one-way analysis of variance fitting routine increased the number of proteins detected at the membrane for two independent populations of Origin 7.0. The data were from a value of 4.5 Ϯ 0.5% (n ϭ 25) in the nontransfected cell considered statistically significant at p Ͻ 0.01
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