Pclamp 10 software suite

Mature brown adipocytes were isolated and attached to coverslips as described above. Whole cell voltage-clamp recordings were performed at room temperature in an external solution containing 145 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 10 mM HEPES (pH 7.4). Recording electrodes (2–5 MΩ) were filled with an internal solution containing 140 mM KCl, 2 mM MgCl₂, 10 mM HEPES, 1 mM EGTA, 4 mM Mg²⁺-ATP, 0.3 mM Na⁺-GTP, and 0.1 mM Na2hosCre (pH 7.2). For voltage-clamp recordings, currents were elicited by 2 s polarizing steps from a holding potential of −60 mV to test potentials ranging from −70 mV to 50 mV in 20 mV increments to study the current-voltage relationship, using an Axopatch 200B amplifier (Axon Instruments). For current-clamp recordings, membrane potential was determined by averaging 50s before NE treatment, 600s during NE treatment, and 120s after NE wash off, respectively. To study the acid-sensitivity, brown adipocytes were differentiated in vitro and attached to coverslips as described above. Currents in response to the 30 mV voltage step were compared at pH 6.4, 7.4, and 8.4 using a perfusion needle. Data were low-pass filtered at 2 kHz, digitized at 20 kHz, and analyzed using the pCLAMP 10 software suite (Molecular Devices).

Whole cell patch-clamp recordings were performed at room temperature in an external solution containing 145 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 10 mM HEPES (pH 7.4). Recording electrodes (2–5 MΩ) were filled with an internal solution containing 135 mM K-Gluconate, 10 mM KCl, 1 mM MgCl₂, 5 mM EGTA, and 10 mM HEPES (pH 7.2). For voltage-clamp recordings, currents were elicited by 3 s polarizing steps from a holding potential of −80 mV to test potentials ranging from −80 mV to 50 mV in 10 mV increments to study the current-voltage relationship, using an Axopatch 200B amplifier (Axon Instruments). For current-clamp recordings, membrane potential was hold at −65 mV and a depolarizing current ramp (0 to 200 pA in one second) was applied to induce action potentials, or hold at around −50 mV to monitor spontaneous firing. Only neurons displaying mature morphology and big sodium currents (> 1nA) were chosen. Data were low-pass filtered at 2 kHz, digitized at 20 kHz, and analyzed using the pCLAMP 10 software suite (Molecular Devices).