2.3 | Patch clamp recordings with bupivacaine enantiomers and ‘side pocket´ mutants
HEK-293 cells were transfected with Kv1.5 cloned in pcDNA3.1 as previously described (Arias, Guizy et al. , 2007; Gonzalez, Navarro-Polanco et al. , 2002). The intracellular pipette filling solution contained (mM): K-aspartate 80, KCl 42, phosphocreatine 3, KH2PO4 10, MgATP 3, HEPES-K 5, and EGTA-K 5, and was adjusted to pH 7.25 with KOH. The bath solution contained (mM): NaCl 145, KCl 4, CaCl2 1.8, MgCl2 1.0, HEPES-Na 10 and glucose 10, and was adjusted to pH 7.40 with NaOH. Both bupivacaine enantiomers were dissolved as stock solutions in deionized MilliQ water at a concentration of 100 mM. Kv1.5 currents were recorded at room temperature (21-23ºC) using the whole-cell patch-clamp technique (Hamill, Marty et al. , 1981) with an Axopatch 200B patch-clamp amplifier and a Digidata 1440A A/D converter (Molecular Devices, California, USA). Currents were filtered at 2 kHz (4-pole Bessel filter) and sampled at 4 kHz. Micropipettes were pulled from borosilicate glass capillary tubes (Narishige, GD-1, Tokyo, Japan) on a programmable horizontal puller (Sutter Instruments Co., California, USA) and heat-polished with a microforge (Narishige, Japan). Micropipette resistance was 2-4 MΩ. Capacitance and series resistance compensation were optimized, and 80% compensation of effective access resistance was usually obtained. Cells were held at −80 mV and 250 ms pulses between -80 mV and +60 mV (in 10 mV steps) were applied at a frequency of 0.1 Hz in order to avoid accumulation of inactivation. Deactivating tail currents were recorded at −40 mV. The IC50 and Hill coefficients, nH , were obtained from fitting the fractional block at various S- or R-bupivacaine concentrations to a Hill plot. The degree of stereoselectivity ”θ” was calculated as fold-difference of half maximal inhibitory concentrations between S-bupivacaine and R-bupivacaine.