Oc 725c

The WT rat TRPV1 and TRPV2 channels in the pcDNA3.1 vector were kindly provided by Dr. David Julius (UCSF) (Caterina et al., 1997 (link); Caterina et al., 1999 (link)) and subcloned into the pGEM-HE vector (Liman et al., 1992 (link)). Mutations were introduced into rTRPV1 using a two-step PCR mutagenesis technique and the resulting constructs were verified by sequencing. All channel constructs were expressed in Xenopus oocytes and studied following 1–4 days incubation after cRNA injection (incubated at 17°C in 96 mM NaCl, 2 mM KCl, 5 mM HEPES, 1 mM MgCl₂ and 1.8 mM CaCl₂, 50 μg/ml gentamycin, pH 7.6 with NaOH) using the two-electrode voltage-clamp recording technique (OC-725C, Warner Instruments, Hamden, CT) with a 150 μl recording chamber. Data were filtered at 1–3 kHz and digitized at 20 kHz using pClamp software (Molecular Devices, Sunnyvale, CA). Microelectrode resistances were 0.1–1 MΩ when filled with 3 M KCl. For recording macroscopic TRP channel currents, the external recording solution contained (in mM): 100 KCl, 10 HEPES, pH 7.6 with KOH. All experiments were performed at room temperature (~22°C).

To measure voltage dependent Ci-VSP phosphatase activities, cRNA of the WT or mutant Ci-VSP were co-injected with that of Kir3.2d, Gβ, and Gγ into oocytes (0.1–0.2: 0.05: 0.05: 0.05 ng/nL, 50 nL injection volume). The injected oocytes were incubated for 2–3 days at 18°C in ND96 solution (5 mM Hepes, 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, and 1 mM MgCl₂ (pH 7.5), supplemented with gentamycin and pyruvate). The macroscopic current was recorded under a two-electrode voltage clamp using an Oocyte Clamp amplifier (OC-725C: Warner Instruments, USA). Stimulation, data acquisition, and analysis were performed on a Macintosh computer using an ITC-16 AD/DA converter and Pulse software (HEKA Electronik, Germany). Intracellular glass microelectrodes were filled with 3 M KCl. The microelectrode resistances ranged from 0.5 to 1.0 MΩ, and the ND96 solution was utilized as a bath solution. Leak subtraction by the P over N protocol was not performed, except for the measurement of ‘sensing’ currents.

Xenopus laevis oocytes were surgically removed and gently shaken for 60 min in a solution of 82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 5 mM HEPES and 2 mg/mL collagenase. A rat TRPV1 construct (generously provided by D. Julius, UCSF) was cloned into the pGEM-HE vector, and used to generate cRNA. The cRNA was then injected into oocytes, which were then incubated for 1–3 days at 17°C in ND-96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 5 mM HEPES, 1 mM MgCl₂ and 50 μg/mL gentamycin, titrated to pH 7.6 with NaOH). TRPV1 activity was recorded under voltage clamp using a two-electrode voltage clamp (OC-725C; Warner Instruments) in a 150-μL recording chamber. The recorded data were filtered at 1 kHz and digitized at 5 kHz using a digidata analog/digital converter and pClamp software (Molecular Devices). Microelectrode resistances were 0.1–1 MΩ when filled with 3 M KCl. The external recording solution contained 115 mM NaCl, 2.5 mM KCl, 1.5 mM MgCl₂ and 10 mM HEPES, titrated to pH 7.4 with NaOH. All experiments were performed at room temperature (~22°C).

Oocytes expressing mRNA during the incubation period were used to observe the potential interaction between the drug and ligand using TEVC (OC-725C; Warner Instruments, Hamden, CT, USA) and Digidata (1322A; Molecular Devices, Sunnyvale, CA, USA). TEVC has voltage and current electrodes that maintain a stable potential and deliver transmembrane potential and large currents. A voltage clamp amplifier transmits large currents of command potential, and Digidata converts the analog signal of the amplifier to enable reading. A computer converts output amplitude of voltage clamp amplifiers into input digital converter using pClamp 10 software (Axon Instruments, Union City, USA). Voltage and current electrodes were filled with 3 M KCl (0.3–0.7 MΩ), and the analysis was conducted at −70 mV holding potential. The oocytes were placed in a chamber and exposed to ND96 solution at a rate of 2 mL per minute. In electrophysiological experiments, the voltage ramp recording was performed at room temperature, and −100 to +80 mV potential was applied to study the current and voltage relationship. Inward peak traces expressed TRPV1, and voltage ramp traces were converted to a suitable value through Clampfit 9.0 (Molecular Devices, San Jose, CA, USA) [37 ,38 (link)].

The voltage clamp recording of Xenopus oocytes was performed as previously described^{38 (link),39 (link)} with slight modifications. Adult Xenopus laevis were acquired from Xenopus Aquaculture Materials. The oocytes were extracted from anesthetized Xenopus laevis. cRNA of AVR-14, GLR-1, SOL-1, and STG-1 was synthesized using the mMessage in vitro transcription kit (Ambion). Approximately 50 nL of the cRNA solution (containing 100 ng/µl cRNA) was injected into each oocyte. After 3–4 days of incubation at 17 °C, voltagec clamp measurements were performed using oc-725c (Warner Instruments). The two electrodes were inserted into oocytes, and oocytes were voltage-clamped to −50 to −60 mV in the extracellular solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES, pH 7.2). Glutamate was applied for 20 s at each concentration (0.1, 0.2, 0.5, 1, 2, 5, and 10 mM, respectively) followed by at least 3 min of wash periods using a perista pump. Dataset was analyzed using Clampfit (10.3.2.1).