Patch-clamp electrophysiology
To conduct electrophysiology experiments, the cells were gravity
perfused with extracellular solution composed of (in mM): 145 NaCl, 5
KCl, 2 CaCl2, 1 MgCl2, 10 D-glucose, and
10 HEPES. The osmolarity was 315 mOsM and the pH was adjusted to 7.4
with NaOH. The flow rate was 1.5 ml/min and was delivered by means of a
polyethylene tube with an inner diameter of 0.58 mm. Patch electrodes
were pulled from borosilicate glass capillaries (Kimbal Chase, cat.
#3400-99) using a P97 pipette puller (Sutter Instruments, Novato CA,
USA). The electrodes had resistances between 1.5 and 3.0 MΩ when filled
with an internal electrode solution (in mM: 145 K-glutamate, 10 NaCl, 1
MgCl2, 10 D-glucose, and 10 HEPES; pH adjusted to 7.2
with KOH; observed osmolarity 322 mOsM). To initiate whole-cell
recordings, a stock solution of 0.5 mg/ml amphotericin B was prepared
daily in dimethysulfoxide. Five μl of this stock solution was added to
500 μl of intracellular solution, ultrasonicated, and used to back fill
the patch electrodes. Experiments were performed under a sodium lamp for
light at 25-27ºC.
A HEKA EPC10 amplifier (HEKA Electronik, Lambrect, Germany) controlled
by PatchMaster software was used to record agonist-evoked responses. The
signals were sampled at 10 kHz and filtered at 1 kHz through a Bessel
filter. The average plasma membrane capacitance (Cm) of
the cells was 5.7 ± 0.7 pF (n=13) for human and 5.6 ± 0.3 pF (n=26) for
rat chromaffin cells, both compensated electronically. Series
resistances ranged between 7 and 15 MΩ and was compensated
electronically by 60-80% in the voltage-clamp mode. In the
current-clamp mode, the resistance was compensated by ≥80% using the
bridge balance feature of PatchMaster.
Agonists were applied in two ways depending on the type of experiment.
For current-clamp experiments, a Picospritzer III (General Valve Corp.,
Fairfield, NJ, USA) was used to apply ACh to the cell by means of
pressure ejection (15 psi) through a glass capillary pipette (World
Precision Instruments, Sarasota, FL, USA; cat. 1B200F-4) with 1-2 µm
diameter tips and filled with extracellular solution containing 300 μM
ACh. For agonist studies in the voltage-clamp mode, a multi barrel
pipette was constructed using polyethylene tubing with an inner diameter
of 0.4 mm. A single polyethylene tube with an inner diameter of 0.28 mm
was used for the outlet. The flow rate of the perfusion system was
approximately 850 μl/min. The agonist pulses were controlled by a valve
controller triggered by the amplifier. For the agonist
concentration-response experiments, 500 ms pulses of agonist were
applied every 3 min. The cells were first stimulated with 300 μM ACh
until steady baseline responses were achieved, and then different
agonists of interest were applied in ascending concentrations. Agonist
responses were normalized to those obtained by 300 μM ACh in the same
cell.
In the triple-step protocol and the recording of nicotinic currents,
each condition was repeated three times and the average value was
obtained. Also, agonists were applied in a different order each time.