Gc150tf 10

Electrophysiological recordings were taken from nAChR-expressing oocytes by two-electrode voltage clamp using a Geneclamp 500 voltage clamp amplifier (Axon instruments, USA). An oocyte was placed in the perfusion chamber using a plastic Pasteur pipette and the bath was perfused (~5 mL/min) with fresh Frog Ringer solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂ and 5 mM HEPES, pH 7.5). Microelectrodes were pulled from borosilicate glass capillaries (Harvard GC150TF-10) using a programmable micropipette puller (Sutter P97, USA), and they had resistances between 0.5 and 2.5 MΩ when filled with 3 M KCl. The oocyte was voltage-clamped at holding potentials (V_H) between −60 and −100 mV. ACh was consistently used as the agonist, and it was applied without or together with PhTX analogues via an 8-channel perfusion system (Automate, USA) for 1 min to allow for equilibration of the current. Currents were recorded to a PC via a digidata 1200 analog-to-digital converter (Axon Instuments, USA) using WinEDR v3.2.6 Software (Dr John Dempster, University of Strathclyde, UK).

cRNA-injected oocytes were incubated for 1–3 days. Ionic currents were recorded with a two-electrode voltage clamp using an OC-725C amplifier (Warner Instruments) at room temperature. The bath chamber was perfused with Ca²⁺-free ND96 solution (96 mM NaCl, 2 mM KCl, 2.8 mM MgCl₂, and 5 mM HEPES, pH 7.6) supplemented with 100 µM LaCl₃ to block endogenous hyperpolarization-activated currents (Osteen et al., 2010 (link)). The microelectrodes were drawn from borosilicate glass capillaries (Harvard Apparatus, GC150TF-10) using a P-1000 micropipette puller (Sutter Instrument) to a resistance of 0.2–1.0 MΩ and filled with 3 M KCl. Currents were elicited from the holding potential of –90 mV to steps ranging from –100 to +60 mV in +20 mV steps each for 2 s with 7.5 s intervals for the KCNQ1-KCNE3 complex analyses and for 5 s with 15 s intervals for the KCNQ1-KCNE1 complex analyses. Oocytes with a holding current larger than –0.2 µA at –90 mV were excluded from the analysis. Generation of voltage-clamp protocols and data acquisition were performed using a Digidata 1550 interface (Molecular Devices) controlled by pCLAMP 10.7 software (Molecular Devices). Data were sampled at 10 kHz and filtered at 1 kHz.

Ionic currents were recorded under two-electrode voltage clamp with an OC-725C amplifier (Warner Instruments) at room temperature. The bath chamber was perfused with ND-96 buffer, containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂ and 5 mM HEPES, pH 7.5. The microelectrodes were drawn from borosilicate glass capillaries (Harvard Apparatus, GC150TF-10) using a P-1000 micropipette puller (Sutter Instrument) to a resistance of 0.2–0.5 MΩ and filled with 3 M KCl. Generation of voltage-clamp protocols and data acquisition were performed using a Digidata 1550 interface (Molecular Devices) controlled by the pClampex 10.7 software (Molecular Devices). Data were sampled at 10 kHz and filtered at 1 kHz by the pClampfit 10.7 software (Molecular Devices).