Reverse engineering the L-type Ca2+ channel alpha1c subunit in adult cardiac myocytes using novel adenoviral vectors.
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.
Ganesan
Anand N
AN
Institute of Molecular Cardiobiology, Department of Medicine, The Johns Hopkins University, Baltimore, MD 21205, USA.
O'Rourke
Brian
B
Maack
Christoph
C
Colecraft
Henry
H
Sidor
Agnieszka
A
Johns
David C
DC
D000256:Adenoviridae / Q000235:genetics*; D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D006168:Guinea Pigs; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D032383:Myocytes, Cardiac / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism
D020746:Calcium Channels, L-Type; D021122:Protein Subunits; D011994:Recombinant Proteins; D002118:Calcium
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