Electrophysiology
Two-electrode voltage clamp (TEVC) recordings were performed on X.
laevis oocytes 24-36 h post injection. Preparation of glass pipettes
was as described in Byrt et al., (2017). TEVC experiments were
performed using an Oocyte Clamp OC-725C (Warner Instruments, Hamden, CT,
USA) with a Digidata 1440A data acquisition system interface (Axon
Instruments, Foster City, CA, USA). Injected oocytes were continuously
perfused with solution after being pierced with the voltage and current
electrodes and allowed to stabilise. TEVC was performed in solutions
consisting of 50 mM NaCl (Na50), 100 mM NaCl (‘Na100’) or 100 mM KCl
(‘K100’) in a basal solution (2 mM KCl, 1 mM MgCl2 and 5
mM HEPES, osmolality was adjusted to 240 mosmol.kg-1with D-mannitol) with 50 µM CaCl2 and pH 8.5. For
experiments involving endogenous oocyte kinase stimulation or
inhibition, injected oocytes were incubated prior to TEVC in Low
Na+ Ringers (described previously) supplemented with 1
mM 8-Br-cAMP (Sigma (St Louis, MO, USA), #B5386), or 1 mM 8-Br-cGMP
(Sigma, #B1381) or 10 µM H7 dihydrochloride (Sigma, #17016) from
concentrated stocks dissolved in water. Steady–state currents were
recorded starting from –40 mV holding potential for 0.5 s and ranging
from 40 mV to –120 mV with 20 mV decrements for 0.5 s before following
a –40 mV pulse for another 0.5 s. Ionic conductance was calculated by
taking the slope of a regression of the linear region across the
reversal potential (–60 mV to +40 mV). TEVC recordings were analysed
with CLAMPEX 9.0 software (pClamp 9.0 Molecular Devices, CA, USA).