4.3. Two Electrode Voltage Clamp Recordings

Two-electrode voltage clamp recordings were used to record currents from AQP-expressing and control oocytes at room temperature. Capillary glass electrodes (1–3 MΩ) were filled with 1 M KCl. Recordings were performed in isotonic saline containing 100 mM NaCl, 2 mM KCl, 5 mM HEPES, and calculated amounts of CaCl₂ buffered with 20 mM EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) to achieve the desired final concentration of free calcium, calculated using the online application (Available online: http://maxchelator.stanford.edu/CaEGTA-NIST.htm, accessed on 31 October 2017). The membrane permeable cyclic GMP analog, CPT-cGMP ((Rp)-8-(para-chlorophenylthio)-cGMP; Sigma-Aldrich) was applied as a bolus into the extracellular bath to achieve a final concentration of 10 μM. Conductance responses were monitored through the experiments by repeated steps to +40mV (800-ms duration) every 6 s from a holding potential of −40 mV, using a GeneClamp amplifier and Clampex 9.0 software (pClamp 9.0 Molecular Devices, Sunnyvale, CA, USA). Data were filtered at 2 kHz and stored to a hard disk for analysis. Quantitative values of the magnitude of relative outward rectification were calculated by standardizing outward current amplitude (at +60 mV) to inward current amplitude (at −80 mV). The AQP1 ion channel blocker AqB011 was custom synthesized by Gary Flynn (Spacefill Enterprises, Bozeman, MT, USA) as described previously [19]. Statistical analyses were done with one-way ANOVA with Bonferroni post hoc tests; p values are indicated in the figure legends.