Xenopus oocyte expression

Oocytes were obtained from wild-type South African clawed frogs (Xenopus Express, Brooksville, FL) following a protocol approved by the Institutional Animal Care and Use Committee of UConn Health. Capped cRNA of lgc-46 was synthesized using a T3 mMessage mMachine Kit (Life Technologies, Carlsbad, CA, USA), and injected into defolliculated oocytes (∼50 nl per oocyte at 1.0 ng nl⁻¹) using a Drummond Nanoject II injector (Drummond Scientific, Broomall, PA, USA). Two-electrode whole-cell recordings were performed 4–5 days after cRNA injection using an oocyte clamp amplifier (OC-725C, Warner Instruments, Hamden, CT, USA) and the Clampex software. The holding voltage was −60 mV. Data were filtered at 1 kHz and sampled at 10 kHz. In each experiment, a single oocyte was placed in an oocyte perfusion chamber (AutoMate Scientific, Berkeley, CA, USA) containing ND96 solution (compositions in mM: 96 NaCl, 2.0 KCl, 1.8 CaCl₂, 1.0 MgCl₂, 5.0 HEPES, pH 7.2). Candidate agonists were perfused into the oocyte perfusion chamber through a micro-manifold (AutoMate Scientific). An 8-channel perfusion controller (Valvelink8.2, AutoMate Scientific) was used for perfusion with its valves controlled by pClamp.