TY - JOUR
T1 - Reverse engineering the L-type Ca2+ channel α1c subunit in adult cardiac myocytes using novel adenoviral vectors
AU - Ganesan, Anand N.
AU - O'Rourke, Brian
AU - Maack, Christoph
AU - Colecraft, Henry
AU - Sidor, Agnieszka
AU - Johns, David C.
N1 - Funding Information:
This work was supported by NIH R01-HL61711 (B. O’R.), NIH R01-NS044541 (D.C.J.), an AHA predoctoral fellowship 0315387U (A.G.), and Deutsche Forschungsgemeineschaft MA 2528/1.1 (C.M.). Under a licensing agreement between Excigen, Inc. and the Johns Hopkins University, Dr. Johns is entitled to a share of royalty and milestone payments received by the University on sales of products described in this article. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.
PY - 2005/4/8
Y1 - 2005/4/8
N2 - The α1c subunit of the cardiac L-type Ca2+ channel, which contains the channel pore, voltage- and Ca2+-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca2+ 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 α1c gene cassettes into the adenovirus backbone. Wild-type (α1c-wt) and mutant (α1c-D-) Ca2+ channel adenoviruses were constructed. The α1c-D- contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both α1c-wt and α1c-D- expressed robustly in A549 cells (peak L-type Ca2+ current (ICaL) at 0 mV: α1c-wt -9.94 ± 1.00 pA/pF, n = 9; α 1c-D- -10.30 pA/pF, n = 12). ICaL carried by α1c-D- was markedly less sensitive to nitrendipine (IC 50 17.1 μM) than α1c-wt (IC50 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 μM nitrendipine blocked only 51 ± 5% (n = 9) of ICaL in α1c-D--expressing myocytes, in comparison to 86 ± 8% (n = 9) of ICaL in control myocytes. Moreover, in 20 μM nitrendipine, calcium transients could still be evoked in α1c-D--transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca2+ release. These α1c adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca2+ channel regulation in the native myocyte background.
AB - The α1c subunit of the cardiac L-type Ca2+ channel, which contains the channel pore, voltage- and Ca2+-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca2+ 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 α1c gene cassettes into the adenovirus backbone. Wild-type (α1c-wt) and mutant (α1c-D-) Ca2+ channel adenoviruses were constructed. The α1c-D- contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both α1c-wt and α1c-D- expressed robustly in A549 cells (peak L-type Ca2+ current (ICaL) at 0 mV: α1c-wt -9.94 ± 1.00 pA/pF, n = 9; α 1c-D- -10.30 pA/pF, n = 12). ICaL carried by α1c-D- was markedly less sensitive to nitrendipine (IC 50 17.1 μM) than α1c-wt (IC50 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 μM nitrendipine blocked only 51 ± 5% (n = 9) of ICaL in α1c-D--expressing myocytes, in comparison to 86 ± 8% (n = 9) of ICaL in control myocytes. Moreover, in 20 μM nitrendipine, calcium transients could still be evoked in α1c-D--transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca2+ release. These α1c adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca2+ channel regulation in the native myocyte background.
KW - Adenovirus
KW - Dihydropyridine
KW - Excitation-contraction coupling
KW - Gene transfer
KW - Ion channel
UR - http://www.scopus.com/inward/record.url?scp=14644439236&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=14644439236&partnerID=8YFLogxK
U2 - 10.1016/j.bbrc.2005.02.015
DO - 10.1016/j.bbrc.2005.02.015
M3 - Article
C2 - 15737650
AN - SCOPUS:14644439236
SN - 0006-291X
VL - 329
SP - 749
EP - 754
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -