The Study Of Acid-induced Chloride Currents In Nasopharyngeal Carcinoma Cells

Objective: Acid-activated chloride currents have been reported in several normal cells and mayplay important roles in regulation of cell function. The extracellular pH of tumor cells is lowerthan normal cells, but the effects of extracellular acidification on chloride channels, thecharacteristics of the acid-sensitive current and the molecular identity of the channel that mediatethe current are still unclear in tumor cells. In this study, the activation and properties of theacid-induced chloride current and the possible candidates of the acid-activated chloride channelwere investigated in human nasopharyngeal carcinoma cells (CNE-2Z).Methods: Whole-cell patch clamp technique was used to record the chloride current innasopharyngeal carcinoma cells (CNE-2Z). The effects of extracellular acidification wereobserved by changing the pH in bath solutions. Hypertonic bath solutions, anionic substitutionand Cl-channel blockers were used to observe the physiological and pharmacologicalcharacteristics of the acid induced chloride current. RT-PCR and Western blot techniques wereused to analyze the expression of the ClC chloride channel families in nasopharyngeal carcinomacells. Specific siRNAs were employed to knock down the expression of various chloridechannels, and were used to identify the candidate protein of the acid-induced chloride channeltogether with the patch clamp technique.Results:1. A chloride current was activated when extracellular pH was reduced to6.6from7.4.The current was weakly outward–rectified and was suppressed by hypertonicity-induced cellshrinkages and by the chloride channel blockers5-nitro-2-3-phenylpropylamino benzoic acid(NPPB), tamoxifen and4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid disodium salt hydrate(DIDS). The permeability sequence of the channel to anions was I-> Br-> Cl-> gluconate.2. The isotonic bath solution with pH5.8could not activate a chloride current. A furtherdecrease of extracellular pH to5.8from6.6inhibited the acid (pH6.6)-induced chloride current.3. Among the ClC families, ClC-3ClC-5and ClC-7mRNA were expressed in CNE-2Zcells, but ClC-5presented a weak expression.4. Knock-down of ClC-3expression with ClC-3siRNA prevented the activation of theacid-induced current. While knock-down of ClC-7expresion with ClC-7siRNA showed noobvious effect on this current. Conclusion: The acid-induced chloride current is expressed in CNE-2Z cells. The chloridechannel mediating the current is volume-sensitive and shares various properties with thevolume-sensitive chloride channel. ClC-3is a candidate of the channel protein that mediates orregulates the acid-activated chloride current in nasopharyngeal carcinoma cells.