Article
the perforated whole-cell patch-clamp technique in order to minimize
current run-down and preserve the intracellular milieu. Amphotericin B
(Sigma A9528) was initially dissolved in DMSO (20 mg ml−1) and used in
the pipette solution at a final concentration of 200 μg ml−1. The tip of the
pipette was filled with amphotericin-free solution containing 80 mM
caesium gluconate, 40 mM CsCl, 10 mM HEPES, 10 mM BAPTA, 1 mM
MgCl 2 and 1 mM Mg-ATP, pH adjusted to 7.2 with CsOH. The pipette was
backfilled with 125 mM CsCl, 10 mM HEPES, 4 mM CaCl 2 , 1 mM MgCl 2
pH 7.2 and CsOH containing amphotericin at 200 μg ml−1. CaCl 2 (4 mM)
was added to the patch electrode solution to enable the detection of
conversion from perforated to ruptured configuration. The external
solution contained 130 mM tetraethylammonium methanesulfonate,
10 mM HEPES, 1 mM MgCl 2 , 10 mM (with Rad expression) or 2 mM
(without Rad expression) BaCl 2 , 5 mM glucose. For experiments with
HEK293T cells, in addition to step protocols, we used a ramp protocol
with a 200-ms voltage ramp from −60 mV to +60 mV (0.6 V s−1) applied
every 10 s to monitor the I–V relationship. All experiments were per-
formed at room temperature, 22 ± 1 °C. Cells were selected on the basis
of co-transfection of a vector containing GFP in the absence of Rad, or
of a vector containing GFP-conjugated Rad. For both cardiomyocytes
and HEK293T cells, cells without a stable baseline (possibly due to run-
down or run-up) were not studied.
The voltage-steps protocol used in cardiomyocytes studies evalu-
ated Ipeak = Ipeak (V), which was recalculated in CLAMPFIT to G = G(V)
as G = I/(V − Erev). For HEK cell experiments, we used a ramp protocol
(Extended Data Fig. 8b). The experimental records were I = I(t), where
t = time. We transformed these records to I = I(V), which was then fur-
ther transformed to G = G(V) in CLAMPFIT (Extended Data Fig. 8b).
The parameters of voltage-dependent activation were obtained using
a modified Boltzmann distribution. A nonlinear fit with Boltzmann
sigmoidal function was carried out in Prism (GraphPad), with a good-
ness of fit (R^2 ) of more than 0.99.
Single-channel patch-clamp electrophysiology
Cell-attached single-channel recordings were performed at room
temperature as described^71 ,^72. Patch pipettes (5–10 MΩ) were pulled
from ultra-thick-walled borosilicate glass (BF200-116-10, Sutter Instru-
ments), and coated with Sylgard. Currents were filtered at 2 kHz. The
pipette solution contained 140 mM tetraethylammonium methane-
sulfonate; 10 mM HEPES; 40 mM BaCl 2 ; at 300 mOsm l−1, adjusted with
tetraethylammonium methanesulfonate; and pH 7.4, adjusted with
tetraethylammonium hydroxide. To maintain the membrane potential
at 0 mV, the bath contained 132 mM potassium glutamate, 5 mM KCl,
5 mM NaCl, 3 mM MgCl 2 , 2 mM EGTA, 10 mM glucose, 20 mM HEPES; at
300 mOsm l−1, adjusted with glucose; and pH 7.4, adjusted with sodium
hydroxide. Cell-attached single-channel currents were measured dur-
ing 200-ms voltage ramps between −80 mV and +70 mV (portions
between −50 mV and +40 mV are displayed and analysed). For each
patch, we recorded 80–120 sweeps with a repetition interval of 10 s.
The sample size for the Po–V relationships are as follows:α1C, n = 10
(3 transfections), 933 sweeps; α1C plus PKA, n = 5 (2 transfections), 450
sweeps; Rad, n = 5 (3 transfections), 372 sweeps; Rad plus PKA, n = 8 (3
transfections), 527 sweeps; 4SA mutant Rad, n = 6 (4 transfections),
388 sweeps; and 4SA mutant Rad plus PKA, n = 5 (3 transfections), 439
sweeps.
The modal analyses are histograms of single-trial average Po values
obtained from one-channel patches. For Po–V analysis, we conserva-
tively corrected for shifts in voltage. The numbers of sweeps are as
follows: α1C alone: 281 sweeps (from three one-channel patches); Rad,
372 sweeps (from five one-channel patches); and Rad plus PKA, 311
sweeps (from three one-channel patches).
Flow-cytometric FRET two-hybrid assay
For flow-cytometric FRET assays, HEK293 cells (ATCC CRL1573) were
cultured in 12-well plates and transfected with polyethylenimine (PEI)
25 kDa linear polymer (Polysciences number 2396602). Briefly, 1.5 μg
of cerulean (Cer)- and venus (Ven)-tagged cDNA pairs were mixed
together in 100 μl of serum-free DMEM media and 6 μl of PEI was
added to each sterile tube. FRET experiments were performed one
day post-transfection. The protein-synthesis inhibitor cycloheximide
(100 μM) was added to cells 2 h before experimentation to halt syn-
thesis of new fluorophores, in order to allow existing fluorophores
to fully mature.
For FRET measurements, we used an LSR II (BD Biosciences) flow
cytometer, equipped with 405-nm, 488-nm and 633-nm lasers for exci-
tation and 18 different emission channels. Forward- and side-scatter
signals were detected and used to gate for single and healthy cells. To
determine FRET efficiency, we measured three distinct fluorescence
signals: first, SCer (corresponding to emission from the cerulean tag) is
measured through the BV421 channel (excitation, 405 nm; emission,
450/50); second, SVe n (corresponding to emission from the venus tag)
is measured via the FITC channel (excitation, 405 nm; dichroic, 505LP;
emission, 525/50); and third, SFRET (corresponding to FRET emission) is
measured via the BV510 channel (excitation, 405 nm; dichroic, 505LP;
emission, 525/50). These raw fluorescence measurements are subse-
quently used to obtain Vendirect (venus emission due to direct excita-
tion), Cerdirect (cerulean emission due to direct excitation), and VenFRET
(venus emission due to FRET excitation). Flow-cytometric signals were
collected at a medium flow rate (2,000 to 8,000 events per second).
Fluorescence data were exported as FCS 3.0 files for further processing
and analysis using custom MATLAB functions (MathWorks).
For each experimental run on the flow cytometer, we performed
several control experiments. First, the background fluorescence level
for each fluorescent channel (BGCer, BGVe n and BGFRET) was obtained by
measuring fluorescence from cells exposed to PEI without any fluoro-
phore-containing plasmids. Second, cells expressing the Ven fluoro-
phore alone were used to measure the spectral crosstalk parameter
RA1, corresponding to bleed-through of Ven fluorescence into the FRET
channel. Third, cells expressing the Cer fluorophore alone were used
to measure spectral crosstalk parameters RD1 and RD2, corresponding
to bleed-through of Cer fluorescence into the FRET and Ven channels
respectively. Fourth, FRET measurements also require determination of
instrument-specific calibration parameters gVe n/gCer and fVe n/fCer, which
are respectively ratios of fluorescence excitation and emission for Ven
to Cer fluorophores. These parameters also incorporate fluorophore-
dependent aspects, including molar extinction (for g) and quantum
yield (for f), as well as instrument-specific parameters, including laser
power, attenuation by optical components, and photodetection, ampli-
fication and digitization of fluorescence. To determine these param-
eters, we used Cer–Ven dimers with four different linker lengths (5, 32,
40 and 228). Fifth, coexpression of Cer and Ven fluorophores provided
estimates for concentration-dependent collisional FRET.
In our experiments, RA1 was approximately 0.11, RD1 approximately
2.8, and RD2 approximately 0.006. We observed only minor day-to-day
variation in these measurements. For each cell, spectral cross-talk was
subtracted as follows: Cerdirect = RD1 × SCer; Vendirect = RA1 × (SVe n – RD2 × SCer);
and VenFRET = SFRET – RA1 × (SVe n – RD2 × SCer) – RD1 × SCer.
Following spectral unmixing, we obtained gVe n/gCer and fVe n/fCer from
data for Cer–Ven dimers by determining the slope and intercept for
the following linear relationship:g
gf
fVen
Cer=×Ven
CerFRET −
directCer
Vendirect
directVen
CerFor typical experiments, gVe n/gCer = 0.0194 and fVe n/fCer = 2.3441. Hav-
ing obtained these calibration values, we computed donor-centric
FRET efficiencies as:E =Ven
Ven+×CerD f
fFRET
FRETdirectVen
Cer