Electrophysiological recordings of TRPC6 currents

TRPC6 currents were recorded from human embryonic kidney 293 (HEK293) cells or L6 cells expressing recombinant TRPC6 with or without sKlotho proteins as previously described (6, 7). Cells expressing TRPC6 were treated with or without sKlotho or recombinant carbohydrate-binding module (CBM) (at indicated concentration) in serum-containing medium overnight. Bacterial fusion protein of CBM containing aa 121–305 of Streptococcus pneumoniae neuraminidase A (NanA) sialidase was prepared and purified as previously described (18). Currents were recorded by voltage-clamp in ruptured whole-cell mode with an Axopatch 200B patch-clamp amplifier and Pulse software (Molecular Devices, Sunnyvale, CA, USA). Cells were held at 0 mV membrane potential and stimulated with repetitive ascending ramp pulse from −100 mV to +100 mV for 400 ms every 10 s. The pipette and bath solution for recording HEK293 cells contained (in mM) 140 CsCl, 1 MgCl₂ 1.5 CaCl₂, 2 ATP-Mg, 5 EGTA, 10 HEPES (pH 7.2 with CsOH) and 140 NaCl, 0.5 EGTA, 10 HEPES, 10 glucose, 10 mannitol (pH 7.4 with NaOH), respectively. The resistance of electrodes containing pipette solution was 1.5–3 MΩ. TRPC6 currents were activated by bath application of oleoyl-acetyl-glycerol (OAG; 100 μM), low pass filtered, at 2 kHz and sampled every 0.1 ms (10 kHz). At the end of experiments, the bath solution was replaced by a solution containing nonpermeant cation N-methyl-d-glucamine to assess TRPC6-mediated inward currents. Data acquisition and analysis were performed using the pClamp v.9.2 program (Molecular Devices) and Prism v.3.0 software (GraphPad Software, La Jolla, CA, USA). Results shown are repeated at least 3 times with similar results.