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

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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

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“…Each data point was reported as the mean of individual measurements Ϯ S.E. as described elsewhere (38,46,50). The free energy of activation was calculated using the midpotential of activation.…”

Section: Methodsmentioning

confidence: 99%

“…Functional Expression of Ca V 3.2-Oocytes were obtained from female Xenopus laevis clawed frogs as described previously (38,39,46). Oocytes were injected with 46 or 4.6 nl of a solution containing 1 g/l of cRNA coding for the Ca V 3.2 WT or mutant and incubated for 2 to 5 days after RNA injection.…”

Section: Methodsmentioning

confidence: 99%

“…Steady-state inactivation was determined using a one-pulse protocol with a 2-s conditioning pulse of varying amplitude followed by a test pulse at the voltage yielding the peak current and was applied at a frequency of 0.02 Hz. Normalized currents obtained at the test pulse (I/I max ) were fitted with a standard Boltzmann equation as described elsewhere (38,46,50). Recovery from inactivation was studied by using a paired-pulse protocol.…”

Section: Methodsmentioning

confidence: 99%

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Cooperative Activation of the T-type CaV3.2 Channel

Demers-Giroux

Bourdin

Sauvé

et al. 2013

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Background:The low activation threshold of T-type Ca V 3.2 channels is central to neuronal rhythmogenesis. Results: The S4-S5 linker of Domain II is functionally coupled with Domains II and III during channel activation. Conclusion: Activation of Ca V 3.2 requires a specific interaction between adjacent domains. Significance: Disrupting this protein interface could be a pharmacological strategy to decrease Ca 2ϩ influx in neuronal pathologies.

“…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%

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

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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.