Identification of glycosylation sites essential for surface expression of the Caᵥα2δ1 subunit and modulation of the Cardiac Caᵥ1.2 channel activity

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 Ca2+ channel complexes. CaVα2δ1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type CaV1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant CaVα2δ1 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 CaVα2δ1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the CaVα2δ1-mediated increase in the peak current density and voltage-dependent gating of CaV1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored CaVα2δ1 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 CaVα2δ1 as well as the CaVα2δ1-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 CaVα2δ1, and furthermore that N-glycosylation of CaVα2δ1 is essential to produce functional L-type Ca2+ channels

T-tubule localization of the inward-rectifier K+ channel in mouse ventricular myocytes: a role in K+ accumulation

The properties of the slow inward ‘tail currents’ (Itail) that followed depolarizing steps in voltage-clamped, isolated mouse ventricular myocytes were examined. Depolarizing steps that produced large outward K+ currents in these myocytes were followed by a slowly decaying inward Itail on repolarization to the holding potential. These currents were produced only by depolarizations: inwardly rectifying K+ currents, IK1, produced by steps to potentials negative to the holding potential, were not followed by Itail.For depolarizations of equal duration, the magnitude of Itail increased as the magnitude of outward current at the end of the depolarizing step increased. The apparent reversal potential of Itail was dependent upon the duration of the depolarizing step, and the reversal potential shifted to more depolarized potentials as the duration of the depolarization was increased.Removal of external Na+ and Ca2+ had no significant effect on the magnitude or time course of Itail. BaCl2 (0.25 mm), which had no effect on the magnitude of outward currents, abolished Itail and IK1 simultaneously.Accordingly, Itail in mouse ventricular myocytes probably results from K+ accumulation in a restricted extracellular space such as the transverse tubule system (t-tubules). The efflux of K+ into the t-tubules during outward currents produced by depolarization shifts the K+ Nernst potential (EK) from its ‘resting’ value (close to −80 mV) to more depolarized potentials. This suggests that Itail is produced by IK1 in the t-tubules and is inward because of the transiently elevated K+ concentration and depolarized value of EK in the t-tubules.Additional evidence for the localization of IK1 channels in the t-tubules was provided by confocal microscopy using a specific antibody against Kir2.1 in mouse ventricular myocytes