Roles Of ClC-3Chloride Channels And IK1Potassium Channels In Cell Volume Regulation And Migration

Objective:1)To investigate the roles of ClC-3chloride channels and IK1potassiumchannels in basal cell volume maintenance and hypotonicity-induced regulatory volume decrease(RVD) in human nasopharyngeal carcinoma CNE-2Z cells and human hepatocellular carcinomacells with high metastatic potential (MHCC97-H).2)To study the dynamic activities and roles ofClC-3and IK1channels in RVD, and the effects of hypotonicity-induced H+secretion and pHchanges on the activities of ClC-3and IK1channels.3)To investigate the roles of IK1potassiumchannels in cell migration, and the spacial and polarized distribution and activities of IK1channels in the processes of cell volume regulation and migration.Methods: The whole-cell patch clamp technique was used to record the chloride andpotassium currents. ClC-3siRNA or IK1siRNA were transfected into cells, and the expressionlevels of ClC-3or IK1proteins were detected by Western blotting. The changes of cell volumeswere determined by image analysis using Scion image software. The Non-invasive micro-testtechnique was used to record the spatiotemporal dynamics of K~+, Cl~-and H+transmembranetransports in intact cells. The extracellular pH (pH_o) near the cell membrane was recorded usingthe H~+selective microelectrode, and intracellular pH (pH_i) was detected using the pH sensitivedye BCECF. IK1channels were subcloned in-frame into enhanced green fluorescent protein(EGFP) fusion vectors. The red fluorescent probe DiI was used to label the cell membrane. Thesubcellular distribution and transport dynamics of EGFP-IK1under isotonic and hypotonicconditions were investigated using a confocal laser scanning microscope. Chemokines wereapplied to induce cell migration. Scratch assay was used to detect the cell migration ability.Results:1) The background chloride and potassium currents recorded by the patch clamptechnique were inhibited by the cell shrinkage induced by hypertonic challenges. Transfection ofCNE-2Z cells with ClC-3siRNA or IK1siRNA knocked down expression of ClC-3or IK1proteins, and attenuated the background chloride or potassium current. Transfection with ClC-3siRNA or IK1siRNA increased basal cell volumes under isotonic conditions. Furthermore, thepurinergic P2Y receptor antagonist RB2increased cell volumes under isotonic conditions with orwithout the existance of extracellular free Ca~2+. Blockage of P2Y receptors by RB2, depletion ofextracellular ATP with apyrase or inhibition of cytoplasm ATP release from maxi-anionchannels by gadolinium chloride decreased the background chloride currents. Extracellularapplications of ATP in micromolar scales activated a chloride current which can be inhibited byhypertonic solutions. Extracellular ATP could also reverse the action of gadolinium chloride.2) Using non-invasive micro-test technique, we proved that transport of K~+and Cl~-was mainly carried by channels in RVD and was uncoupled in CNE-2Z cells, with the transient K~+efflux activated earlier and sustained Cl~-efflux activated later. Hypotonic challenges decreasedintracellular pH (pH_i), and activated a proton pump-dependent H+efflux, resulting in a decline ofextracellular pH (pH_o). Modest decreases of pH_oinhibited the volume-activated K~+outflow andRVD, but not the Cl~-outflow, while inhibition of H+efflux or increase of pH_obuffer abilitypromoted K~+efflux and RVD. Whole-cell patch clamp recordings demonstrated that thevolume-activated K~+or Cl~-currents had similar pharmacological properties with the ion effluxes,respectively. Transfection of IK1siRNA or ClC-3siRNA inhibited hypotonic induced K~+and Cl~-transmembrane transports.3) Stable background potassium currents and hypotonicity-activated potassium currentswere recorded in MHCC97-H cells, and both could be inhibited by the potassium channelblocker clotrimazole. Transfection of cells with IK1siRNA knocked down expression of IK1proteins, attenuated the background and hypotonic activated potassium currents, and increasedcell volumes under isotonic conditions. IK1channels were subcloned in-frame into pEGFP-C2expression vector, and green fluorescence can be observed by fluorescence imaging in cellstransfected with EGFP fusions of IK1channels (EGFP-IK1). Cells transiently transfected withGFP-IK1exhibited augmented background K~+currents that were blocked by the potassiumchannel blocker clotrimazole. Under isotonic conditions, uneven GFP-IK1fluorescence signalmay be found in cells, and the local volume decrease level was positively correlated with signalintensity. After hypotonic stimulation, the GFP-IK1in cytoplasm was vesicular-like aggregated,transported from cytoplasm onto the cell membrane by exocytosis, resulting in the increaseamount of GFP-IK1proteins in the entire plasma membrane. Scratch assay indicated that bothpotassium channel blocker clotrimazole and IK1siRNA could inhibit cell migration. The K~+, H~+and Ca~2+transmembrane transports were polarized in migrating cells. The K~+influx was detectedin the fronts and efflux in the rears. The H+transports in the fronts and rears were both efflux,with the stronger efflux in the fronts than the rears. Ca~2+influx was detected in the rears, withoutsignificant transmembrane transports recorded in the fronts. The K~+efflux in the rears but not theinflux in the fronts could be inhibited by clotrimazole. IK1channels were concentrated at therears in migrating cells. Gradual increase of EGFP-IK1at the rears was also observed infibronectin induced cell migrations, and increased GFP-IK1fluorescence signal was found in theplasma membrane at the rears. GFP-IK1fluorescence signal was also found concentrated at theruffling leading edge in the fronts of migrating cells. The transport of EGFP-IK1betweencytoplasm and cell membrane was different in the fronts and rears in migrating cells. EGFP-IK1was transported from cytoplasm to cell membrane in the rears, and transported from cell membrane to cytoplasm in the fronts.Conclusion: The present study indicates that ClC-3chloride channels and IK1potassiumchannels play important roles in cell volume regulation in human nasopharyngeal carcinoma(CNE-2Z) cells and human hepatocellular carcinoma (MHCC97-H) cells. Under isotonicconditions, ClC-3chloride channels can be activated by autocrine ATP through purinergicreceptor pathways and may be involved in maintenance of basal cell volume. Inhypotonicity-induced RVD, the differential sensitivity of K~+and Cl~-channels to extracellaracidification uncoupled temporally the K~+and Cl~-transports. IK1proteins are involved in cellmigration. Functional expression or activities of the potassium channels are polarized inmigrating cells. Transition between the cytoplasm and cell membrane and/or diffusion in the cellmembrane may modulate the amount of IK1proteins in entire or local cell membrane, whichmay be responsible for regulation of cell volume and migration. Polarized transmembranetransports of H+and Ca~2+may also regulate cell migration via modulation of IK1channel activityand K~+transports.