Electrophysiological Characterization of TRPV4 Channels

The whole-cell current was measured with an EPC-10 amplifier and PatchMaster Software (HEKA Elektronik, Lambrecht, Germany). Patch electrodes had resistances of 3 to 8 MΩ. TRPV4 currents were recorded continuously at 30-second intervals from a holding potential of 0 mV to a voltage ramp from −100 mV to +100 mV over 100 ms.^{19 (link)} The pipette solution contained (in millimolar) 20 CsCl, 100 Cs⁺-aspartate, 1 MgCl₂, 4 ATP, 0.08 CaCl₂, 10 BAPTA, and 10 HEPES (pH 7.2). The bath solution contained (in millimolar) 150 NaCl, 6 CsCl, 1 MgCl₂, 1.5 CaCl₂, 10 glucose, and 10 HEPES (pH 7.4). All recordings were made before and after the specific TRPV4 channel agonist, GSK1016790A (No. G0798; Sigma, 10 nmol/L)^{20 (link),21 (link)} application. These values were then plotted versus time. The single-channel current was measured with an Axopatch 200B amplifier and pClamp 10 software.^{22 (link)} The pipette solution contained (in millimolar) 140 NaCl, 1 MgCl₂, and 10 HEPES (pH 7.4). The bath solution contained (in millimolar): 135 KCl, 0.8 CaCl₂, 1 MgCl₂, 5 glucose, 5 EGTA, and 5 HEPES (pH 7.3). All recordings were made under control conditions and after treatment with GSK1016790A (10 nmol/L) or the selective TRPV4 channel antagonist, HC067047 (No. SML0143; Sigma, 10 μM).^{23 (link),24 (link)} Experiments were performed at room temperature.