Reverse engineering the L-type Ca2+ channel alpha1c subunit in adult cardiac myocytes using novel adenoviral vectors

Abstract

The alpha(1c) subunit of the cardiac L-type Ca(2+) channel, which contains the channel pore, voltage- and Ca(2+)-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca(2+) channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length alpha(1c) gene cassettes into the adenovirus backbone. Wild-type (alpha(1c-wt)) and mutant (alpha(1c-D-)) Ca(2+) channel adenoviruses were constructed. The alpha(1c-D-) contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both alpha(1c-wt) and alpha(1c-D-) expressed robustly in A549 cells (peak L-type Ca(2+) current (I(CaL)) at 0 mV: alpha(1c-wt) -9.94+/-1.00pA/pF, n=9; alpha(1c-D-) -10.30pA/pF, n=12). I(CaL) carried by alpha(1c-D-) was markedly less sensitive to nitrendipine (IC(50) 17.1 microM) than alpha(1c-wt) (IC(50) 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 microM nitrendipine blocked only 51+/-5% (n=9) of I(CaL) in alpha(1c-D-)-expressing myocytes, in comparison to 86+/-8% (n=9) of I(CaL) in control myocytes. Moreover, in 20 microM nitrendipine, calcium transients could still be evoked in alpha(1c-D-)-transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca(2+) release. These alpha(1c) adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca(2+) channel regulation in the native myocyte background.

Fig. 1

Creation of the fiber deleted ψ5 virus. Schematic showing the two parental viruses that were used to create novel recombination compatible fiber deleted virus, FB6. Purified viral DNA was cut with EcoRI and run on a 0.6% agarose gel. Lane 1, DNA size marker; lane 2, ψ5 DNA; lane 3, ψFB6 DNA; and lane 4, GFP-F DNA. The presence of bands * and ** in ψFB6 is consistent with the formation of a hybrid virus from ψ5 and GFP-F parent viruses.

Fig. 2

α_1c-D- and α_1c-wt adenoviruses are robustly expressed in A549 cells. (A,B) Example records shown for α_1c-wt and α_d1c-D- adenoviruses, each co-expressed in A549 cells with β_2a-GFP and AdDMEcR adenoviruses, following voltage steps from −50 to 0 mV. (C) Dose–response curves for α_1c-D- and α_1c-wt adenoviruses, each co-expressed in A549 cells with β_2a and AdDMEcR adenoviruses, following voltage steps from −50 to 0 mV, in sequentially increasing concentrations of the dihydryopyridine antagonist, nitrendipine.

Fig. 3

α_1c-D- adenoviruses are expressed in cardiac myocytes (A,B): individual current traces obtained before and after the administration of 10 µM nitrendipine, during voltage steps from −50 to 0 mV, in (A) β_2a transduced adult guinea pig cardiomyocytes and (B) α_1c-D- and β_2a transduced cardiomyocytes. (C) Current to voltage relationships plotted for α_{1c- D-} adenovirus, co-expressed in myocytes with β_2a and AdDMEcR adenoviruses (D) dose-response curves for α_1c-D- adenoviruses, each co-expressed in adult guinea pig cardiac myocyte with β_2a and AdDMEcR adenoviruses, following voltage steps from −50 to 0 mV, in sequentially increasing concentrations of the dihydryopyridine antagonist, nitrendipine. As a control, the effect of nitrendipine on myocytes transduced with β_2a and AdDmEcR alone is shown.

Fig. 4

α_1c-D- adenoviruses expressed in cardiac myocytes are able to elicit SR Ca release. Examples of steady state [Ca]_i transients are shown, during stimulation with 50 ms depolarizing steps from −50 to 0 mV, before and after the administration of 20 µM nitrendipine, in (A) β_2a transduced adult guinea pig cardiomyocytes and (B) α_1c-D- and β_2a transduced cardiomyocytes.