A Specific Tryptophan in the I-II Linker Is a Key Determinant of β-Subunit Binding and Modulation in CaV2.3 Calcium Channels

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca 2؉ channel complexes. Ca V ␣2␦1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type Ca V 1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant Ca V ␣2␦1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of Ca V ␣2␦1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the Ca V ␣2␦1-mediated increase in the peak current density and voltage-dependent gating of Ca V 1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored Ca V ␣2␦1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of Ca V ␣2␦1 as well as the Ca V ␣2␦1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of Ca V ␣2␦1, and furthermore that N-glycosylation of Ca V ␣2␦1 is essential to produce functional L-type Ca 2؉ channels.The regulation of Ca 2ϩ influx in cardiac cells is critical to the generation of the force necessary for the myocardium to meet the physiological needs of the body (1). In resting cells, intracellular free ionized Ca 2ϩ is maintained at a low concentration (high nanomolar range) by the concerted action of mechanisms that prevent Ca 2ϩ entry, promote its extrusion (mostly via the Na ϩ /Ca 2ϩ exchanger), and ensure its storage in the sarcoplasmic reticulum (2). Ca 2ϩ entry is mediated mainly by the cardiac L-type Ca 2ϩ channel, which is central to the initiation of excitation-contraction coupling via Ca 2ϩ -induced Ca 2ϩ release from the sarcoplasmic reticulum. Regulation of the L-type Ca 2ϩ current has profound physiological significance. Indeed, alterations in density or the activation/inactivation gating of L-type Ca 2ϩ channels have been implicated in a variety of cardiovascular diseases (3, 4), including cardiac arrhythmias such as atrial fibrillation (5-8), heart failure (9, 10), and ischemic heart disease (10). The molecular mechanisms underlying changes in the activity of the L-type Ca 2ϩ channel remain under study for most pathologies.The L-type Ca V 1.2 channel belongs to the molecular family of high voltage-activated Ca V channels. High voltage-activated Ca V 1.2 channels are hetero-oligo...

Section: Discussionmentioning

confidence: 99%

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

Tétreault¹,

Bourdin²,

Briot³

et al. 2016

“…The voltage-dependent calcium channel (VDCC) 1 represents one key pathway that regulates the entry of extracellular calcium into electrically excitable cells. VDCC are multimeric proteins composed of the transmembrane pore-forming Ca V ␣1, the disulfide-linked dimer Ca V ␣2␦, the intracellular Ca V ␤ subunits (␤1-␤4), and in some cases the Ca V ␥ subunit (2).…”

mentioning

confidence: 99%

The C-terminal Residues in the Alpha-interacting Domain (AID) Helix Anchor CaVβ Subunit Interaction and Modulation of CaV2.3 Channels

Berrou

Dodier

Raybaud

et al. 2005

The alpha-interacting domain (AID) in the I-II linker of high voltage-activated (HVA) Ca 2؉ channel ␣1 subunits binds with high affinity to Ca V ␤ auxiliary subunits. The recently solved crystal structures of the AIDCa V ␤ complex in Ca V 1.1/1.2 have revealed that this interaction occurs through a set of six mostly invariant residues Glu/Asp 6 (9). Ca V ␤ subunit modulation of LVA T-type (Ca V 3.1-3.3) channels has yet to be reported (10).Ca V ␤ subunits increase peak current density, in part by recruiting the Ca V ␣1 subunit to the plasma membrane (11)(12)(13)(14), by hyperpolarizing the voltage dependence of activation and inactivation, and by increasing the channel opening probability at the single channel level (4, 15). Ca V ␤ subunits increase inactivation kinetics in an isoform-specific manner with Ca V ␤3 Ͼ Ca V ␤1b Ͼ Ca V ␤1a Ͼ Ca V ␤4 Ͼ Ͼ Ca V ␤2a (4). The four known auxiliary Ca V ␤ subunits can potentially associate with any of the six Ca V ␣1 pore-forming subunits of HVA VDCC (Ca V 1.1-1.4, Ca V 2.1-2.3) via the alpha interaction domain (AID) located on the I-II linker of the Ca V ␣1 subunit. The AID motif is absent from LVA T-type (Ca V 3.1-3.3) VDCC for which Ca V ␤ subunit modulation has never been reported. About half of the residues of the AID helix (Gln 1 -Gln 2 -X 3 -Glu 4 -X 5 -X 6 -Leu 7 -X 8 -Gly 9 -Tyr 10 -X 11 -X 12 -Trp 13 -Ile 14 -X 15 -X 16 -X 17 -Glu 18 ) are strictly conserved (in bold) among the six Ca V ␣1 subunits but homology was found to be higher within members of the Ca V 1 and Ca V 2 families. Nonetheless, positions 8, 11, and 15 are occupied by residues that could vary even within the same Ca V family (Table I) (where X can be any residue) might not be the unique determinant of the Ca V ␣1-␤ interaction. In vitro binding experiments have revealed the presence of additional interaction sites of lower affinity on the cytoplasmic loops of Ca V 2.1 (16,17) and on the C-terminal of Ca V 2.3 (18) but the AID appears to be the primary high affinity site of interaction (14, 15).

“…This is a surprising observation given that the reverse observation was reported in the Xenopus expression system. AID-deficient Ca V 2.3 channels migrated to the plasma membrane but were not functionally modulated by Ca V ␤ when expressed in Xenopus oocytes (37,38). It certainly raises questions about the transposition of data from Xenopus oocytes to mammalian cells.…”

Section: Discussionmentioning

confidence: 99%

“…Whereas interaction between Ca V ␤ and Ca V ␣1 is required for the trafficking of Ca V ␣1 to the plasma membrane, it remains to be seen whether nanomolar binding of Ca V ␤ to the AID motif of Ca V ␣1 is required to carry both roles. Furthermore, whereas the binding of Ca V ␤ on the AID of Ca V ␣1 has been well characterized (17,22,26,33,37,38), few concur on the GK residues in Ca V ␤ that determine protein density at the plasma membrane and contribute to channel modulation.…”

mentioning

confidence: 99%

See 1 more Smart Citation

A Quartet of Leucine Residues in the Guanylate Kinase Domain of CaVβ Determines the Plasma Membrane Density of the CaV2.3 Channel

Shakeri

Bourdin

Demers-Giroux

et al. 2012

Background: Ca V ␤ subunits stimulate cell surface expression of Ca V 2.3 channels. Results:Of 33 positions and domains tested, leucine mutants in the guanylate kinase domain of Ca V ␤3 decreased significantly the surface protein density of Ca V 2.3. Conclusion: Leucine residues are responsible for the functional modulation by Ca V ␤. Significance: A quartet of leucine residues forms the hydrophobic pocket surrounding the ␣-interacting domain of Ca V 2.3.