Biomedical Engineering Reference
In-Depth Information
used to derive membrane capacitance from currents elicited by - 10 and + 10 mV
square pulses while holding the cell at RMP. Series resistance was not
compensated for.
I
K1
was elicited by applying 1 s square test pulses ranging
between - 130 and + 50 mV from a holding potential of -40 mV. Steady state
currents at the end of the pulse were normalized to membrane capacitance
and plotted against test pulse potential. Offline analysis was done using
MacDaq 8.0 (kindly provided by Dr A.C.G. van Ginneken) and R 2.0.1 [13].
Experiments were done at 20° C on a Nikon Diaphot 300 inverted
microscope. Extracellular buffer used was a modified Tyrode's solution,
containing (in mM) NaCl 140, KCl 5.4, CaCl
2
1.8, MgCl
2
1, HEPES 15,
NaHCO
3
35, glucose 6, pH 7.20/NaOH. Gap-junctional coupling was inhibited
by applying 4 mM Halothane dissolved in extracellular bu
er at the interface
between NCM and HEK-KWGF cells with an additional micropipette.
Pipette bu
er contained (in mM) potassium gluconate 125, KCl 10, HEPES 5,
EGTA 5, MgCl
2
2, CaCl
2
0.6, Na
2
ATP 4, pH 7.20/KOH. Patch pipettes were
pulled on a Narishige PC-10 puller and fire-polished. When filled with pipette
bu
er, the pipette resistance ranged between 2 and 5 M
ȍ
. Liquid junction
potential was calculated using Clampex (Molecular Devices Corp, Sunnyvale,
CA, USA) and used for offline correction.
Beating frequency was determined by counting the number of beats for a
period of 30 s under visual inspection. For each condition, at least six
independent NCM or NCM/HEK-KWGF clusters were analyzed.
3 Results
3.1 Validation of
I
K1
Expressing HEK-KWGF Cells
To create a co-culture system able to test electrotonic coupling of
I
K1
expressing
cells with spontaneous active NCMs, we first created cells which were in
principle able to couple with cardiomyocytes and that expressed functional
I
K1
.
Therefore, HEK-293 cells were stably transfected with a murine Kir2.1-GFP
fusion protein expression construct. Upon G418 based selection and FACS
assisted enrichment, the resulting HEK-KWGF cells were further characterized.
Western blot analysis demonstrated the expression of the Kir2.1-GFP fusion
protein in stably transfected cells, while no signal was observed in maternal
HEK-293 cells or NCM (Fig. 1a). Analysis of the gap-junction protein Cx43
expression demonstrated the presence of Cx43 in HEK-293 and HEK-KWGF
cells; however, the levels were fairly low when compared to Cx43 expression in
rat NCMs (Fig. 1a). In contrast, Cadherin expression, required for mechanical
interaction between the two cell types, displayed less di
erence in expression
level between the cell types. Epifluorescence microscopy illustrated the presence
of a strong GFP signal at the cell borders of HEK-KWGF cells, and therefore
Kir2.1 channels appear to be expressed at the plasma membrane (Fig. 1b).
Subsequently, electrophysiological measurements indeed validated the presence
