Two-electrode voltage clamping

Ecocyte Bioscience supplied defolliculated oocytes which were promptly resuspended in modified Barth’s solution containing the following: 88 mM NaCl, 1 mM KCl, 0.82 mM MgSO₄, 0.33 mM Ca(NO₃)₂·4H₂O, 0.41 mM CaCl₂·2H₂O, 2.4 mM NaHCO₃, and 5 mM Hepes supplemented with amikacin 250 (mg/liter) and gentamycin (150 mg/liter). Xenopus laevis oocytes were then injected with either hP2X₁ (50 nl of 100 ng/μl), hP2X₂ (50 nl of 50 ng/μl), hP2X₃ (50 nl of 400 ng/μl), hP2X₄ (50 nl of 200 ng/μl), hP2X₇ (50 nl of 10 ng/μl), or rP2X₇ (50 nL of 20 ng/μl or 50 ng/μl) mRNA created from linearized full-length, wild-type or mutant pcDNA 3.1x according to the protocol provided in the mMESSAGE mMACHINE kit (Invitrogen). After injection, the oocytes were allowed to express protein for ~20 hours before recording was performed. Oocytes expressing hP2X₄ were allowed to express protein for ~48 hours.

All TEVC data were acquired with an Ooctye Clamp OC-725C amplifier, pClamp 8.2 software, and a gravity-fed RSC-200 Rapid Solution Changer that flowed buffer at ~5 ml/min. For recording, Sutter filamented glass (10 cm in length with an inner diameter of 0.69 mm and an outer diameter of 1.2 mm) was used to impale oocytes and clamp the holding voltage at −60 mV. Recordings for hP2X₁, hP2X₂, hP2X₇, and rP2X₇ were performed in buffer with the following: 100 mM NaCl, 2.5 mM KCl, 0.1 mM EDTA, 0.1 mM flufenamic acid, and 5 mM Hepes at pH 7.4. Recordings for hP2X₃ and hP2X₄ were performed in buffer with the following: 10 mM Hepes (pH 7.4), 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, and 10 mM glucose. For TEVC experiments with P2X₇, oocytes expressing hP2X₇ or rP2X₇ were facilitated with 100 μM ATP before recording the final data.

To evaluate the inhibitory response of an antagonist for a dilution series, an initial excitatory signal was evoked at a subtype-specific concentration of ATP. Next, the antagonist was applied at a test concentration for 60 s and then coapplied at the same test concentration of the antagonist with the identical subtype-specific concentration of ATP. The antagonized signal was then normalized against the preceding excitatory signal evoked by ATP for each individual oocyte tested. The pre- and post-antagonist signals were evoked by 10 μM ATP for hP2X₁, 10 μM ATP for hP2X₂, 1 μM ATP for hP2X₃, 50 μM ATP for hP2X_4, and 100 μM ATP for P2X₇. For the K297V mutation in rP2X₇, pre- and post-antagonist signals were evoked by 300 μM ATP. Each dataset was then fit to a nonlinear regression named “[inhibitor] vs. response – Variable slope (four parameters)” in GraphPad Prism 9 to produce a sigmoidal curve and afford an IC₅₀ value. Each singular condition was used to generate an average which is reported plus or minus its SD between three discrete trials.

After testing an oocyte with a brief 100 μM ATP excitatory current, an antagonist is applied for 40, 120, or 240 s at IC₁₀₀ calculated from IC₅₀ values. The oocyte is then treated with 20 s of antagonist at the IC₁₀₀ plus 100 μM ATP to see complete antagonism. This is followed by 120 s of washing with buffer, ending with a 20-s reapplication of 100 μM ATP. The signal after the reapplication of ATP was then compared to the initial excitatory current to afford a percentage of recovery of activation after complete antagonism. Experiments were run in triplicate. Trials were averaged and errors were reported as SD. The recovery of the receptor was tested after the application of an IC₁₀₀ of each drug at the following concentrations: A438079 at 6.6 μM, A839977 at 1 μM, AZD9056 at 3 μM, GSK1482160 at 81 μM, methyl blue at 108 μM, and JNJ47965567 at 1.3 μM.