© 2000 American Heart Association, Inc.
Cellular Biology |
Ca2+ Channel Modulation by Recombinant Auxiliary ß Subunits Expressed in Young Adult Heart Cells
From the Program in Molecular and Cellular Systems Physiology (S.-k.W., H.M.C., C.D.D., B.Z.P., R.Z., D.T.Y.), Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Md; Department of Medicine (T.A.K.), Howard Hughes Medical Institute, Duke University, Durham, NC; and Department of Biochemistry and Molecular Biology (T.B.R.), University of Maryland School of Medicine, Baltimore, Md.
Correspondence to David T. Yue, MD, PhD, Ross Building, Room 713, Johns Hopkins University School of Medicine, 720 Rutland Ave, Baltimore, MD 21205. E-mail dyue{at}bme.jhu.edu
Abstract—L-type Ca2+
channels contribute importantly to the normal excitation-contraction
coupling of physiological hearts, and to the
functional derangement seen in heart failure. Although Ca2+
channel auxiliary ß1–4 subunits are among the strongest
modulators of channel properties, little is known about their role in
regulating channel behavior in actual heart cells. Current
understanding draws almost exclusively from heterologous expression of
recombinant subunits in model systems, which may differ from
cardiocytes. To study ß-subunit effects in the cardiac
setting, we here used an adenoviral-component gene-delivery strategy to
express recombinant ß subunits in young adult ventricular
myocytes cultured from 4- to 6-week-old rats. The main results were the
following. (1) A component system of replication-deficient adenovirus,
poly-L-lysine, and expression plasmids encoding ß
subunits could be optimized to transfect young adult myocytes with 1%
to 10% efficiency. (2) A reporter gene strategy based on green
fluorescent protein (GFP) could be used to identify
successfully transfected cells. Because fusion of GFP to ß subunits
altered intrinsic ß-subunit properties, we favored the use of a
bicistronic expression plasmid encoding both GFP and a ß subunit. (3)
Despite the heteromultimeric composition of L-type
channels (composed of 
1C, ß, and 2
),
expression of recombinant ß subunits alone enhanced Ca2+
channel current density up to 3- to 4-fold, which argues that ß
subunits are "rate limiting" for expression of current in heart.
(4) Overexpression of the putative "cardiac" ß2a
subunit more than halved the rate of voltage-dependent inactivation at
+10 mV. This result demonstrates that ß subunits can tune
inactivation in the myocardium and suggests that other ß
subunits may be functionally dominant in the heart. Overall, this study
points to the possible therapeutic potential of ß subunits to
ameliorate contractile dysfunction and excitability in heart
failure.
Key Words: Ca2+ channel • ß subunit • adenovirus • gene delivery
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