Outwardly rectifying swelling-activated chloride conductance (ICl,Swell) in rabbit heart plays a critical role in cardioprotection following ischemic preconditioning (IP). But the functional characterization and molecular basis of this chloride conductance in rabbit heart ventricular myocytes is not clear. Candidate chloride channel clones (e.g. ClC-2, ClC-3, ClC-4 and ClC-5) were determined using RT-PCR and Western blot analysis. Whole cell ICl,Swell was recorded from isolated rabbit ventricular myocytes using patch clamp techniques during hypo-osmotic stress. The inhibitory effects of 4,4' isothiocyanato-2,2-disulfonic acid (DIDS), 5-nitro-2(3-phenylroylamino) benzoic acid (NPPB) and indanyloxyacetic acid 94 (IAA-94) on ICl,Swell were examined. The expected size of PCR products for ClC-2, ClC-3 and ClC-4 but not for ClC-5 was obtained. ClC-2 and ClC-3 expression was confirmed by automated fluorescent DNA sequencing. RT-PCR and Western blot showed that ClC-4 was expressed in abundance and ClC-2 was expressed at somewhat lower levels. The biological and pharmacological properties of I(Cl,Swell), including outward rectification, activation due to cell volume change, sensitivity to DIDS, IAA-94 and NPPB were identical to those known properties of ICl,Swell in exogenously expressed systems and other mammals hearts. It was concluded that ClC-3 or ClC-4 might be responsible for the outwardly rectifying part of ICl,Swell and may be the molecular targets of cardioprotection associated with ischemic preconditioning or hypo-osmotic shock.
Biophysics/methods ; Chlorides/*chemistry ; Chlorides/metabolism ; DNA Primers/chemistry ; Electrophysiology/methods ; Gene Expression Regulation ; Glycolates/pharmacology ; Heart Ventricles/*cytology ; Ischemic Preconditioning ; Muscle Cells/*cytology ; Osmosis ; Sequence Analysis, DNA

OBJECTIVETo re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a (hASIC1a) current in HEK293 cells with a modified perfusion methods.

METHODSWith cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASIC1a currents evoked by low pH external solution.

RESULTSUsing cell floating method, the amplitude of hASIC1a currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is (21+/-5) ms and (270+/-25) ms, respectively. Inactivation time constants are (496+/-23) ms and (2284+/-120) ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC1a [IC50 is (3.4+/-1.1) micromol/L and (2.4+/- 0.9) micromol/L, respectively]. Both recording methods have similar pH activation EC50 (6.6+/-0.6, 6.6+/-0.7, respectively).

CONCLUSIONASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC1a current was re-confirmed and the biophysical and pharmacological properties of hASIC1a channel in HEK293 cells were precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.

Acid Sensing Ion Channels ; Amiloride ; pharmacology ; Biophysics ; instrumentation ; methods ; Cell Culture Techniques ; instrumentation ; methods ; Cell Line ; Cell Membrane ; chemistry ; drug effects ; metabolism ; Culture Media ; chemistry ; pharmacology ; Extracellular Fluid ; chemistry ; metabolism ; Humans ; Hydrogen-Ion Concentration ; drug effects ; Membrane Potentials ; drug effects ; physiology ; Nerve Tissue Proteins ; chemistry ; drug effects ; metabolism ; Neuropharmacology ; instrumentation ; methods ; Patch-Clamp Techniques ; instrumentation ; methods ; Perfusion ; instrumentation ; methods ; Sodium Channel Blockers ; pharmacology ; Sodium Channels ; chemistry ; drug effects ; metabolism ; Time Factors