Biomedical Engineering Reference
In-Depth Information
HCN2
HCN2 + HA-MiRP1
0
-1000
-2000
-3000
012345
012345
Time (sec)
*
1000
*
800
*
*
*
600
400
200
0
-110
-100
-90
-80
-70
-60
-50
Voltage (mV)
Fig. 3.
Co-expression of [hyperpolarization activated, cyclic nucleotide gated (HCN)] HCN2
and HA-tagged MiRP1 in neonatal ventricular myocytes alters current magnitude and kinetics.
Top
Representative HCN2 currents recorded from a myocyte co-infected with adenoviruses
expressing HCN2 and green fluorescent protein (GFP) (as a control;
left
) and one co-infected
with adenoviruses expressing HCN2 and HA-tagged MiRP1 (
right
). Test voltages were -45, -
65, -85, -105 mV; the holding potential was -35 mV. Bottom Effect of HA-tagged MiRP1 co-
expression on kinetics of HCN2 activation and deactivation. Activation (
circles
) and
deactivation (
squares
) time constants are plotted as a function of voltage for HCN2 and MiRP1
co-expression (
unfilled symbols
) and compared to those for HCN2 and GFP co-expression
(
filled symbols
). The solid lines are the best-fit curves to the equation IJ = 1/(
A
1×exp(-
V
/
B
1) +
A
2×·exp(
V
/
B
2)) where IJ is the activation or deactivation kinetic time constant;
A
1,
A
2,
B
1, and
B
2 are calculated fitting parameters.
Asterisk
indicates significant differences (reprinted by
permission from reference [11]).
[11]. In other experiments, we have explored a point mutation in murine HCN2
(E324A) that was reported to exhibit both faster kinetics and a more positive
activation relation than HCN2 [2], both characteristics that should enhance
pacemaking. In preliminary data, we found that while the mutation did indeed show
these preferred characteristics in myocyte cultures, it also expressed less well than the
wild-type HCN2 gene, resulting in significantly reduced current magnitude [6].
Finally the HCN2 construct was responsive both to the acceleratory effect of beta-
adrenergic catecholamine and to the deceleratory effects of acetylcholine.
Encouraged by the implications of the cell culture work, we then proceeded to test
proof-of-concept by injecting a small quantity of HCN2 and GFP genes in an
adenoviral vector into canine left atrium [10]. We permitted the animals to recover
