Mature female Xenopus laevis frogs (Xenopus I, Ann Arbor, MI, USA) were kept in dechlorinated tap water at 19–21 °C and fed with beef liver at least twice a week. Clusters of oocytes were removed surgically under tricaine (Sigma, St. Louis, MO, USA) anesthesia (0.15%). Individual oocytes were manually dissected in a solution containing (in mM): NaCl, 88; KCl, 1; NaHCO3, 2.4; MgSO4, 0.8; HEPES, 10 (pH 7.5) and stored 2–7 days in modified Barth’s solution containing (in mM): NaCl, 88; KCl, 1; NaHCO3, 2.4; Ca(NO3)2, 0.3; CaCl2, 0.9; MgSO4, 0.8; HEPES, 10 (pH 7.5), supplemented with sodium pyruvate 2 mM, penicillin 10,000 IU/L, streptomycin 10 mg/L, and gentamicin 50 mg/L [29 (link),30 (link)]. Oocytes were placed in a 0.2 mL recording chamber and superfused at a constant rate of 5–7 mL/min. The bathing solution consisted of (in mM): NaCl, 95; KCl, 2; CaCl2, 2; and HEPES 5 (pH 7.5). The cells were impaled at the animal pole with two glass microelectrodes filled with 3 M KCl (1–3 MΩ). The oocytes were routinely voltage clamped at a holding potential of −20 mV using a GeneClamp-500 amplifier (Axon Instruments Inc., Burligame, CA, USA). Current responses were digitized by an A/D converter and analyzed using pClamp 6 (Axon Instruments Inc., Burligame, CA, USA) run on an IBM/PC or directly recorded on a Gould 2400 rectilinear pen recorder (Gould Inc., Cleveland, OH, USA). Current-voltage characteristics were studied using 1 s voltage steps (–120 to 20 mV). All chemicals and drugs including glibenclamide, MB HCl, BAPTA-AM, and 8-Br-cAMP were from Sigma (St. Louis, MO, USA). BAY 58-2667 (dissolved in DMSO) was obtained from Tocris-Bio-Techne (Minneapolis, MN, USA). Drugs were applied externally by addition to the superfusate. Procedures for the injections of BAPTA (50–70 nL, 100 mM) were described earlier in detail [31 (link)].
For inside-out patch experiments, oocytes were chemically defolliculated by collagenase 1A treatment (Sigma, St. Louis, MO, USA; 2 mg/mL, 2 h) and injected with 2 ng cRNA encoding Kir6.2ΔC26 mutant. After 2 days of incubation in modified Barth’s solution, oocytes were placed in hypertonic solution containing 200 mM K+-aspartate at pH 7.0, and the vitelline layer was removed with sharpened watchmaker’s forceps. The pipette (external) solution contained (in mM) 140 KCl, 1.2 MgCl2, 2.6 CaCl2, and 10 HEPES (pH 7.4). Intracellular (bath) solution contained (in mM) 107 KCl, 10 EGTA, 2 MgCl2, 1 CaCl2, and 10 HEPES (pH 7.2 with KOH; final K+ ≅140). Patch pipettes had a resistance of 250–400 kΩ when filled with the pipette solution. Currents were recorded at 20–22 °C from giant inside-out patches using Axopatch 200B amplifier (Axon Instruments Inc., Burligame, CA, USA) at a holding potential of 0 mV, sampled at a rate of 2 kHz and filtered at 1 kHz. Currents were evoked by 2 s voltage ramps from −100 mV to +100 mV at a pulse frequency of 0.5 Hz. In each inside-out patch, the efficacy of 1 mM K2ATP to block the KATP current was tested before applying MB. Leak currents recorded at 10 mM K2ATP at the end of the experiments were subtracted from recordings. The recording chamber had a volume of 250 μL and the perfusion rate was 2 mL/min. Due to light sensitivity of MB, experiments were conducted in the dark and MB containers and perfusion lines kept in the dark by aluminum foils.
Statistical significance at the level of 0.05 was analyzed using the Student’s t-test, paired t-test or ANOVA. Concentration-response curves were obtained by fitting the data to the logistic equation,
where x and y are concentration and response, respectively, Emax is the maximal response, EC50 is the half-maximal concentration, and n is the slope factor. For data analysis, calculations, and fits of the data, the computer software Origin 8.5 (Microcal Software-OriginLab Corp., Northampton, MA, USA) was used.
For radioligand binding experiments, follicle-enclosed oocytes were suspended in 300 mL of buffer containing 50 mM HEPES, 300 mM sucrose and 1 mM EDTA at 4 °C on ice as described earlier [32 (link),33 (link)]. Oocytes were homogenized using a motorized Teflon homogenizer (six strokes, 15 s each at high speed). This was followed by sequential centrifugations at 1000× g (10 min) and 10,000× g (20 min); each time the pellet was discarded and the supernatant was used for the subsequent step. The final centrifugation was at 60,000× g for 25 min. The microsomal pellet, which contains the membranes of follicular cells [34 (link)], was resuspended in 50 mM HEPES buffer and used for the binding studies.
The radioligand binding experiments were carried out at room temperature (20–22 °C) for 1 h. Oocyte membranes were incubated in 1 mL of 50 mM HEPES, pH 7.5, at a protein concentration of 200–500 μg/mL. [3H]glibenclamide was dissolved in ethanol/dimethyl sulphoxide (1:1). For each experiment, freshly made glibenclamide solution was used. For the analysis, calculations, nonlinear curve fitting and regression fits of the radioligand binding data, the computer software Origin 8.5 (Microcal Software-OriginLab Corp., Northampton, MA, USA) was used.
For inside-out patch experiments, oocytes were chemically defolliculated by collagenase 1A treatment (Sigma, St. Louis, MO, USA; 2 mg/mL, 2 h) and injected with 2 ng cRNA encoding Kir6.2ΔC26 mutant. After 2 days of incubation in modified Barth’s solution, oocytes were placed in hypertonic solution containing 200 mM K+-aspartate at pH 7.0, and the vitelline layer was removed with sharpened watchmaker’s forceps. The pipette (external) solution contained (in mM) 140 KCl, 1.2 MgCl2, 2.6 CaCl2, and 10 HEPES (pH 7.4). Intracellular (bath) solution contained (in mM) 107 KCl, 10 EGTA, 2 MgCl2, 1 CaCl2, and 10 HEPES (pH 7.2 with KOH; final K+ ≅140). Patch pipettes had a resistance of 250–400 kΩ when filled with the pipette solution. Currents were recorded at 20–22 °C from giant inside-out patches using Axopatch 200B amplifier (Axon Instruments Inc., Burligame, CA, USA) at a holding potential of 0 mV, sampled at a rate of 2 kHz and filtered at 1 kHz. Currents were evoked by 2 s voltage ramps from −100 mV to +100 mV at a pulse frequency of 0.5 Hz. In each inside-out patch, the efficacy of 1 mM K2ATP to block the KATP current was tested before applying MB. Leak currents recorded at 10 mM K2ATP at the end of the experiments were subtracted from recordings. The recording chamber had a volume of 250 μL and the perfusion rate was 2 mL/min. Due to light sensitivity of MB, experiments were conducted in the dark and MB containers and perfusion lines kept in the dark by aluminum foils.
Statistical significance at the level of 0.05 was analyzed using the Student’s t-test, paired t-test or ANOVA. Concentration-response curves were obtained by fitting the data to the logistic equation,
where x and y are concentration and response, respectively, Emax is the maximal response, EC50 is the half-maximal concentration, and n is the slope factor. For data analysis, calculations, and fits of the data, the computer software Origin 8.5 (Microcal Software-OriginLab Corp., Northampton, MA, USA) was used.
For radioligand binding experiments, follicle-enclosed oocytes were suspended in 300 mL of buffer containing 50 mM HEPES, 300 mM sucrose and 1 mM EDTA at 4 °C on ice as described earlier [32 (link),33 (link)]. Oocytes were homogenized using a motorized Teflon homogenizer (six strokes, 15 s each at high speed). This was followed by sequential centrifugations at 1000× g (10 min) and 10,000× g (20 min); each time the pellet was discarded and the supernatant was used for the subsequent step. The final centrifugation was at 60,000× g for 25 min. The microsomal pellet, which contains the membranes of follicular cells [34 (link)], was resuspended in 50 mM HEPES buffer and used for the binding studies.
The radioligand binding experiments were carried out at room temperature (20–22 °C) for 1 h. Oocyte membranes were incubated in 1 mL of 50 mM HEPES, pH 7.5, at a protein concentration of 200–500 μg/mL. [3H]glibenclamide was dissolved in ethanol/dimethyl sulphoxide (1:1). For each experiment, freshly made glibenclamide solution was used. For the analysis, calculations, nonlinear curve fitting and regression fits of the radioligand binding data, the computer software Origin 8.5 (Microcal Software-OriginLab Corp., Northampton, MA, USA) was used.
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