Transfection and whole-cell patch-clamp recording

Human embryonic kidney 293 (HEK293) cells were cultured and patch clamped as described previously (40). Cells were transfected using FuGENE 6 (Promega, Madison, WI) or X-tremeGENE HP (Roche Diagnostics, Indianapolis, IN) with 0.5 μg GFP and 1 μg CLH-3b cDNAs ligated into pcDNA3.1. Point and deletion mutations and concatamers were generated using QuikChange Lightning Multi Site-Directed mutagenesis kits (Agilent Technologies, Santa Clara, CA). Concatemeric channel constructs were generated by linking two CLH-3b monomers C-terminus to N-terminus via a 19-amino-acid linker with the sequence LRGQDNSADIQHSGGRSSA. All channel constructs were confirmed by DNA sequencing. Experimental protocols were performed on at least two independently transfected groups of cells.

Transfected cells were identified by GFP fluorescence and patch clamped using a bath solution containing 90 mM N-methyl-d-glucamine chloride (NMDG-Cl), 5 mM MgSO₄, 1 mM CaCl₂, 12 mM Hepes free acid titrated to pH 7.0 with CsOH, 8 mM Tris, 5 mM glucose, 90 mM sucrose, and 2 mM glutamine (pH 7.4, 300 mOsm), and a pipette solution containing 116 mM NMDG-Cl, 2 mM MgSO₄, 20 mM Hepes, 6 mM CsOH, 1 mM EGTA, 2 mM ATP, 0.5 mM GTP, and 10 mM sucrose (pH 7.2, 275 mOsm). Patch electrodes were pulled from silanized borosilicate microhematocrit tubes (outer diameter: 1.5 mm), and electrode resistance ranged from 4 to 8 MΩ. Currents were measured with an Axopatch 200A (Axon Instruments, Foster City, CA) patch-clamp amplifier. Electrical connections to the patch-clamp amplifier were made using Ag/AgCl wires and 3 M KCl/agar bridges. Data acquisition and analysis were performed using pClamp 10 software (Axon Instruments).