Catalog #P6181) was added at 1:20 ratio of enzyme to substrate an

Catalog #P6181) was added at 1:20 ratio of enzyme to substrate and incubated at 37 °C for ∼24 h Rat.

The patch clamp method (Hamill et al., 1981) was used to trace and record, ionic currents from heterologous expression systems over-expressing a recombinant channel. GW-572016 cost NaV1.3 channels were expressed in HEK-293 cells as described (Cummins et al., 2001). There is some degree of endogenous expression of NaV channels in HEK cells, but their contribution in comparison to exogenously expressed channels is usually minute (Moran et al., 2000). Rat NaV1.8 channels were expressed in ND7-23 cells by using conventional transient or stable transfections as described (Zhou et al., 2003, John et al., 2004, Jarvis et al., 2007 and Zimmermann et al., 2007). HEK cells stably expressing human NaV1.3, human NaV1.8 and human NaV1.7 channels were purchased from Scottish Biomedical (Glasgow, UK). Human NaV1.5 channels were expressed in HEK-293 cells as described

(Van Bemmelen et al., 2004). The patch clamp set up included amplifier and digitizer (Axopatch 200B and DIGDATA 1322A, FGFR inhibitor Axon instruments, USA), microscope (Nikon ECLIPSE 100) and micromanipulator (MP-225-Sutter Instrument Co., USA). Recording pipettes were pulled from Borosilicate glass tubes (Sutter Instrument co., USA). Cells were always perused with control extracellular solutions and changing to reagents containing solutions was performed using ValveLink 16 (Automate Scientific Inc. Berkeley, USA) and a peristaltic pump (Ismatec, Wertheim, Germany) perfusion system. Intracellular (pipette) solution (in mM): 120 CsF, 10 NaCl, 10 TEA-OH, 1 MgCl2, 1 CaCl2, 11 EGTA, Aspartate 10 HEPES (pH = 7.2 titrated with KOH). The extracellular (bath) solution contained (in mM) 115 NaCl, 20 TEA-OH, 1 MgCl2, 2 CaCl2, 5 glucose, 10 HEPES (pH = 7.4 titrated with NaOH), supplemented with 600 nM TTX when recording rat or human NaV1.8 channel currents. All channels were activated

using the following stimulation protocol: Holding level −100 mV, ramp from −100 to +60 mV (50 ms), delivered every 10 s and recorded at a sampling rate of 10–50 kHz. The ramp protocol is increasingly in use as a quick measure of I–V relationship (see for example Dib-Hajj et al., 2007). Indeed, it is possible that measuring inhibition upon square pulse stimulation, may have yield somewhat different results (maybe shifting the dose response curves). However, the ramp stimulation method has enabled the use of exactly the same stimulation protocol with all channels tested. All chemicals were from Sigma–Aldrich (Rehovot, Israel) apart from TTX from Alomone Labs (Jerusalem, Israel). All results are presented as mean ± standard deviation.

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