Swelling‐induced chloride current in glioblastoma proliferation,migration, and invasion

Glioblastoma (GBM) remains as the most common and aggressive brain tumor. The survival of GBM has been linked to the aberrant activation of swelling‐induced chloride current I_Cl,swell. In this study, we investigated the effects of I_Cl,swell on cell viability, proliferation, and migration in the human GBM cell lines, U251 and U87, using a combination of patch clamp electrophysiology, MTT, colony formation, wound healing assays and Western immunoblotting. First, we showed that the specific inhibitor of I_Cl,swell, DCPIB, potently reduced the I_Cl,swell in U87 cells. Next, in both U87 and U251 cells, we found that DCPIB reduced GBM viability, proliferation, colony formation, migration, and invasion. In addition, our Western immunoblot assay showed that DCPIB‐treated U251 cells had a reduction in JAK2, STAT3, and Akt phosphorylation, thus, suggesting that DCPIB potentially suppresses GBM functions through inhibition of the JAK2/STAT3 and PI3K/Akt signaling pathways. Therefore, the I_Cl,swell may be a potential drug target for GBM.     

Functional ion channels and cell proliferation in 3T3-L1 preadipocytes

Mouse 3T3-L1 preadipocytes are widely used for metabolic study of obesity; however, their cellular physiology is not fully understood. The present study investigates functional ion channels and their role in the regulation of cell proliferation using whole-cell patch voltage-clamp, RT-PCR, Western blot, and cell proliferation assay in undifferentiated 3T3-L1 preadipocytes. We found three types of ionic currents present in 3T3-L1 preadipocytes, including an inwardly-rectifying K(+) current (I(Kir), recorded in 15% of cells) inhibited by Ba(2+), a Ca(2+)-activated intermediate K(+) current (IK(Ca), recorded in 44% of cells) inhibited by clotrimazole (or TRAM-34) as well as a chloride current (I(Cl)) inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in 12% of cells, which can be activated in all cells with hypotonic (0.8 T) insult, implicating a volume-sensitive I(Cl) (I(Cl.vol)). RT-PCR and Western blot analysis revealed the expression of KCa3.1 (for IK(Ca)), Kir2.1 (for I(Kir)), and Clcn3 (for I(Cl.vol)). Blockade of IK(Ca) with TRAM-34 or I(Cl.vol) with DIDS inhibited cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific siRNAs also suppressed cell proliferation. Flow cytometry analysis showed that blockade or silencing of KCa3.1 or Clcn3 channels with corresponding blockers or siRNAs caused an accumulation of cells at the G0/G1 phase. These results demonstrate that three functional ion channel currents, I(KCa), I(Cl.vol), and I(Kir), are heterogeneously present in 3T3-L1 preadipocytes. I(KCa) and I(Cl.vol) participate in the regulation of cell proliferation.     

Apical Ca2+-activated potassium channels in mouse parotid acinar cells

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.     

KCa3.1 channels are involved in the infiltrative behavior of glioblastoma in vivo

Glioblastoma multiforme (GBM) is a diffuse brain tumor characterized by high infiltration in the brain parenchyma rendering the tumor difficult to eradicate by neurosurgery. Efforts to identify molecular targets involved in the invasive behavior of GBM suggested ion channel inhibition as a promising therapeutic approach. To determine if the Ca²⁺-dependent K⁺ channel KCa3.1 could represent a key element for GBM brain infiltration, human GL-15 cells were xenografted into the brain of SCID mice that were then treated with the specific KCa3.1 blocker TRAM-34 (1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole). After 5 weeks of treatment, immunofluorescence analyses of cerebral slices revealed reduced tumor infiltration and astrogliosis surrounding the tumor, compared with untreated mice. Significant reduction of tumor infiltration was also observed in the brain of mice transplanted with KCa3.1-silenced GL-15 cells, indicating a direct effect of TRAM-34 on GBM-expressed KCa3.1 channels. As KCa3.1 channels are also expressed on microglia, we investigated the effects of TRAM-34 on microglia activation in GL-15 transplanted mice and found a reduction of CD68 staining in treated mice. Similar results were observed in vitro where TRAM-34 reduced both phagocytosis and chemotactic activity of primary microglia exposed to GBM-conditioned medium. Taken together, these results indicate that KCa3.1 activity has an important role in GBM invasiveness in vivo and that its inhibition directly affects glioma cell migration and reduces astrocytosis and microglia activation in response to tumor-released factors. KCa3.1 channel inhibition therefore constitutes a potential novel therapeutic approach to reduce GBM spreading into the surrounding tissue.     

Ca2+-activated IK1 channels associate with lipid rafts upon cell swelling and mediate volume recovery

Restoration of cell volume in the continued presence of osmotic stimuli is essential, particularly in hepatocytes, which swell upon nutrient uptake. Responses to swelling involve the Ca2+-dependent activation of K+ channels, which promote fluid efflux to drive volume recovery; however, the channels involved in hepatocellular volume regulation have not been identified. We found that hypotonic exposure of HTC hepatoma cells evoked the opening of 50 pS K+-permeable channels, consistent with intermediate conductance (IK) channels. We isolated from rat liver and HTC cells a cDNA with sequence identity to the coding region of IK1. Swelling-activated currents were inhibited by transfection with a dominant interfering IK1 mutant. The IK channel blockers clotrimazole and TRAM-34 inhibited whole cell swelling-activated K+ currents and volume recovery. To determine whether IK1 underwent volume-sensitive localization, we expressed a green fluorescent protein fusion of IK1 in HTC cells. The localization of IK1 was suggestive of distribution in lipid rafts. Consistent with this, there was a time-dependent increase in colocalization between IK1 and the lipid raft ganglioside GM1 on the plasma membrane, which subsequently decreased with volume recovery. Pharmacological disruption of lipid rafts altered the plasma membrane distribution of IK1 and inhibited volume recovery after hypotonic exposure. Collectively, these findings support the hypothesis that IK1 regulates compensatory responses to hepatocellular swelling and suggest that regulation of cell volume involves coordination of signaling from lipid rafts with IK1 function.     

Voltage-dependent ionic conductances in the human malignant astrocytoma cell line U87-MG

Although the human malignant astrocytoma cell line U87-MG has been used in numerous studies, few findings are available on the properties of its membrane ion conductances. Characterization of the ion channels expressed in these cells will make it possible to study membrane ion conductance changes when a receptor is activated by its ligand. This will help to elucidate the functional properties of these receptors and their signal-transduction pathways in pathophysiological events. This work studied the voltage-dependent ionic conductances of U87-MG cells using the Whole-Cell Recording patch-clamp technique. Six types of voltage-dependent ionic currents were identified: (i) a TEA-, 4-AP- and CTX-sensitive Ca2+-dependent K+ current, (ii) a transient K+ current inhibited by 4-AP, (iii) an inwardly rectifying K+ current blocked by Ba2+ and 4-AP, (iv) a DIDS- and SITS-sensitive Cl- current, (v) a TTX-sensitive Na+ conductance and (vi) a L-type Ca2+ conductance activated by BayK-8644 and inhibited by Ni and the L-type Ca2+ channel inhibitor, nifedipine. In addition, electrical depolarizations elicited inward currents due to voltage-independent, Ca2+-dependent K+ influx against the electrochemical gradient, probably via an ouabain-sensitive Na+-K+ pump.     

Regulation of cell proliferation of human induced pluripotent stem cell-derived mesenchymal stem cells via ether-à-go-go 1 (hEAG1) potassium channel

The successful generation of a high yield of mesenchymal stem cells (MSCs) from human induced pluripotent stem cells (iPSCs) may represent an unlimited cell source with superior therapeutic benefits for tissue regeneration to bone marrow (BM)-derived MSCs. We investigated whether the differential expression of ion channels in iPSC-MSCs was responsible for their higher proliferation capacity than BM-MSCs. The expression of ion channels for K(+), Na(+), Ca(2+), and Cl(-) was examined by RT-PCR. The electrophysiological properties of iPSC-MSCs and BM-MSCs were then compared by patch-clamp experiments to verify their functional roles. Significant mRNA expression of ion channel genes including KCa1.1, KCa3.1, KCNH1, Kir2.1, SCN9A, CACNA1C, and Clcn3 was observed in both human iPSC-MSCs and BM-MSCs, whereas Kir2.2 and Kir2.3 were only detected in human iPSC-MSCs. Five types of currents [big-conductance Ca(2+)-activated K(+) current (BK(Ca)), delayed rectifier K(+) current (IK(DR)), inwardly rectifying K(+) current (I(Kir)), Ca(2+)-activated K(+) current (IK(Ca)), and chloride current (I(Cl))] were found in iPSC-MSCs (83%, 47%, 11%, 5%, and 4%, respectively) but only four of them (BK(Ca), IK(DR), I(Kir), and IK(Ca)) were identified in BM-MSCs (76%, 25%, 22%, and 11%, respectively). Cell proliferation was examined with MTT or bromodeoxyuridine assay, and doubling times were 2.66 and 3.72 days for iPSC-MSCs and BM-MSCs, respectively, showing a 1.4-fold discrepancy. Blockade of IK(DR) with short hairpin RNA or human ether-à-go-go 1 (hEAG1) channel blockers, 4-AP and astemizole, significantly reduced the rate of proliferation of human iPSC-MSCs. These treatments also decreased the rate of proliferation of human BM-MSCs albeit to a lesser extent. These findings demonstrate that the hEAG1 channel plays a crucial role in controlling the proliferation rate of human iPSC-MSCs and to a lesser extent in BM-MSCs.     

Glioma-specific cation conductance regulates migration and cell cycle progression

In this study, we have investigated the role of a glioma-specific cation channel assembled from subunits of the Deg/epithelial sodium channel (ENaC) superfamily, in the regulation of migration and cell cycle progression in glioma cells. Channel inhibition by psalmotoxin-1 (PcTX-1) significantly inhibited migration and proliferation of D54-MG glioma cells. Both PcTX-1 and benzamil, an amiloride analog, caused cell cycle arrest of D54-MG cells in G(0)/G(1) phases (by 30 and 40%, respectively) and reduced cell accumulation in S and G(2)/M phases after 24 h of incubation. Both PcTX-1 and benzamil up-regulated expression of cyclin-dependent kinase inhibitor proteins p21(Cip1) and p27(Kip1). Similar results were obtained in U87MG and primary glioblastoma multiforme cells maintained in primary culture and following knockdown of one of the component subunits, ASIC1. In contrast, knocking down δENaC, which is not a component of the glioma cation channel complex, had no effect on cyclin-dependent kinase inhibitor expression. Phosphorylation of ERK1/2 was also inhibited by PcTX-1, benzamil, and knockdown of ASIC1 but not δENaC in D54MG cells. Our data suggest that a specific cation conductance composed of acid-sensing ion channels and ENaC subunits regulates migration and cell cycle progression in gliomas.     

Fetal calf serum-injected F1 mice spontaneously generate specific anti-parental cytotoxic T lymphocytes in in vitro culture

Spleen cells from adult (BALB/c x AKR/J)F1 mice primed in vivo with fetal calf serum (FCS) can spontaneously generate anti-parental AKR/J cytotoxic T cells (CTL) in a 5-day in vitro culture containing 5% FCS. This response is distinguished by the following features: (i) it is anti-parental but not anti-self, and (ii) it has specificity for the Kk parental determinant as shown by mapping studies on a variety of targets and antiserum-blocking experiments. Although specifically elicited by FCS and mediated by FCS-induced T-helper cells, it is ascertained that this cytotoxicity is not directed against Kk components modified by absorbed FCS as shown by cold-target competition studies. Further experiments involved a comparative investigation of the patterns of lysis of allogenically induced CTL, FCS-induced CTL, and natural killer (NK) cytotoxic activities on tumor cell targets. The resistance of BW 5147 tumor targets to NK- and FCS-induced lysis was found to be dramatically overcome by treatment with mitomycin C, and provides circumstantial evidence for a functional relationship between the FCS-induced anti-parental CTL effectors and NK cells based on the observed similarity in lytic patterns of these two effector types. With reference to the work of other authors, the possibility that hybrid resistance and its possible in vitro counterpart, F1 anti-parental CTL cytotoxicity, and NK activity are mediated by similar or common effector mechanisms is discussed.     

Cyclic AMP-induced K⁺ secretion occurs independently of Cl⁻ secretion in rat distal colon

cAMP induces both active Cl(-) and active K(+) secretion in mammalian colon. It is generally assumed that a mechanism for K(+) exit is essential to maintain cells in the hyperpolarized state, thus favoring a sustained Cl(-) secretion. Both Kcnn4c and Kcnma1 channels are located in colon, and this study addressed the questions of whether Kcnn4c and/or Kcnma1 channels mediate cAMP-induced K(+) secretion and whether cAMP-induced K(+) secretion provides the driving force for Cl(-) secretion. Forskolin (FSK)-enhanced short-circuit current (indicator of net electrogenic ion transport) and K(+) fluxes were measured simultaneously in colonic mucosa under voltage-clamp conditions. Mucosal Na(+) orthovanadate (P-type ATPase inhibitor) inhibited active K(+) absorption normally present in rat distal colon. In the presence of mucosal Na(+) orthovanadate, serosal FSK induced both K(+) and Cl(-) secretion. FSK-induced K(+) secretion was 1) not inhibited by either mucosal or serosal 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34; a Kcnn4 channel blocker), 2) inhibited (92%) by mucosal iberiotoxin (Kcnma1 channel blocker), and 3) not affected by mucosal cystic fibrosis transmembrane conductance regulator inhibitor (CFTR(inh)-172). By contrast, FSK-induced Cl(-) secretion was 1) completely inhibited by serosal TRAM-34, 2) not inhibited by either mucosal or serosal iberiotoxin, and 3) completely inhibited by mucosal CFTR(inh)-172. These results indicate that cAMP-induced colonic K(+) secretion is mediated via Kcnma1 channels located in the apical membrane and most likely contributes to stool K(+) losses in secretory diarrhea. On the other hand, cAMP-induced colonic Cl(-) secretion requires the activity of Kcnn4b channels located in the basolateral membrane and is not dependent on the concurrent activation of apical Kcnma1 channels.     

Exaggerated inflammatory responses mediated by Burkholderia cenocepacia in human macrophages derived from Cystic fibrosis patients

Glioblastoma (GBM) is a highly aggressive cancer type characterized by intense neovascularization. Several lines of evidence indicate that blood clotting enzymes play an important role in the tumor microenvironment, mainly through the activation of protease-activated receptors (PAR). In particular, PAR1 and PAR2 isoforms may activate signal transduction pathways that promote a number of pro-tumoral responses. However, little is known concerning the role of PAR1/PAR2 in GBM progression. In this study, we investigated the expression and function of PAR1 and PAR2 in the human GBM cell lines A172 and U87-MG. We also evaluated the effect of agonist peptides for PAR1 (PAR1-AP) and PAR2 (PAR2-AP) on signaling pathways and the expression of vascular endothelial growth factor (VEGF). Immunoblotting assays showed that A172 and U87-MG constitutively express PAR1 and PAR2. Treatment of GBM cells with PAR1-AP or PAR2-AP enhanced Akt (protein kinase B) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in a time-dependent manner. LY29042 and PD98059, inhibitors of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, decreased PAR-mediated activation of Akt and ERK1/2, respectively. In addition, we observed that PAR2, but not PAR1, activation increased VEGF secretion in U87-MG and A172 cells. Notably, only PD98059 reduced PAR2-mediated VEGF production by GBM cells. Our results suggest that PAR2 modulates VEGF production through the MAPK/ERK1/2 pathway, and not the PI3K/Akt pathway, in human GBM cell lines. Therefore, the PAR2/MAPK signaling axis might be regarded as a relevant target for adjuvant treatment of GBM with a possible impact on tumor angiogenesis.     

Cation selectivity and inhibition of malignant glioma Na+ channels by Psalmotoxin 1

Psalmotoxin 1 (a component of the venom of a West Indies tarantula) is a 40-amino acid peptide that inhibits cation currents mediated by acid-sensing ion channels (ASIC). In this study we performed electrophysiological experiments to test the hypothesis that Psalmotoxin 1 (PcTX1) inhibits Na⁺ currents in high-grade human astrocytoma cells (glioblastoma multiforme, or GBM). In whole cell patch-clamped cultured GBM cells, the peptide toxin quickly and reversibly inhibited both inward and outward current with an IC₅₀ of 36 ± 2 pM. The same inhibition was observed in freshly resected GBM cells. However, when the same experiment was performed on normal human astrocytes, the toxin failed to inhibit the whole cell current. We also determined a cationic selectivity sequence for inward currents in three cultured GBM cell lines (SK-MG-1, U87-MG, and U251-MG). The selectivity sequence yielded a unique biophysical fingerprint with inward K⁺ conductance approximately fourfold greater than that of Na⁺, Li⁺, and Ca²⁺. These observations suggest that PcTX1 may prove useful in determining whether GBM cells express a specific ASIC-containing ion channel type that can serve as a target for both diagnostic and therapeutic treatments of aggressive malignant gliomas. patch clamp; amiloride; ion channels; acid-sensing ion channels     

Immediate early gene IEX-1 induces astrocytic differentiation of U87-MG human glioma cells

The immediate early response gene IEX-1 is involved in the regulation of apoptosis and cell growth. In order to increase the apoptotic sensitivity to chemotherapeutic drugs and gamma-ray, we attempted to establish U87-MG human glioma cell line expressing IEX-1. Unexpectedly, however, transfection of IEX-1 into U87-MG glioma cells resulted in morphological changes to astrocytic phenotype and increase in glial differentiation marker proteins, S-100 and glial fibrillary acidic protein (GFAP). Glial cell differentiation was used to examine in rat C6 glioma cell line, since this cell line express astrocytic phenotypes by increase in intracellular cAMP concentration. Stimulation of human U87-MG glioma cells by membrane-permeable dibutyryl cAMP (dbcAMP) not only elicited their morphological changes but also induced expression of IEX-1 as well as S-100 and GFAP. H89, an inhibitor of protein kinase A (PKA), blocked dbcAMP-induced morphological changes of U87-MG cells and expression of IEX-1. In contrast, morphological changes and expression of S-100 and GFAP induced by IEX-1 were not affected by H89. Morphological changes induced by dbcAMP were totally abolished by functional disruption of IEX-1 expression by anti-sense RNA. These results indicate that IEX-1 plays an important role in astrocytic differentiation of human glioma cells and that IEX-1 functions at downstream of PKA.     

Subcellular distribution of calcium-sensitive potassium channels (IK1) in migrating cells

Cell migration is crucial for wound healing, immune defense, or formation of tumor metastases. In addition to the cytoskeleton, Ca2+ sensitive K+ channels (IK1) are also part of the cellular "migration machinery." We showed that Ca2+ sensitive K+ channels support the retraction of the rear part of migrating MDCK-F cells by inducing a localized shrinkage at this cell pole. So far the molecular nature and in particular the subcellular distribution of these channels in MDCK-F cells is unknown. We compared the effect of IK1 channel blockers and activators on the current of a cloned IK1 channel from MDCK-F cells (cIK1) and the migratory behavior of these cells. Using IK1 channels labeled with a HA-tag or the enhanced green fluorescent protein we studied the subcellular distribution of the canine (cIK1) and the human (hIK1) channel protein in different migrating cells. The functional impact of cIK1 channel activity at the front or rear part of MDCK-F cells was assessed with a local superfusion technique and a detailed morphometric analysis. We show that it is cIK1 whose activity is required for migration of MDCK-F cells. IK1 channels are found in the entire plasma membrane, but they are concentrated at the cell front. This is in part due to membrane ruffling at this cell pole. However, there appears to be only little cIK1 channel activity at the front of MDCK-F cells. In our view this apparent discrepancy can be explained by differential regulation of IK1 channels at the front and rear part of migrating cells.     

General Sensitization of melanoma cells for TRAIL-induced apoptosis by the potassium channel inhibitor TRAM-34 depends on release of SMAC

The death ligand TRAIL represents a promising therapeutic strategy for metastatic melanoma, however prevalent and inducible resistance limit its applicability. A new approach is presented here for sensitization to TRAIL. It is based on inhibition of the membrane potassium channel KCa3.1 (IK1), which serves fundamental cellular functions related to membrane potential. The selective inhibitor TRAM-34 did not induce apoptosis by itself but synergistically enhanced TRAIL sensitivity and overrode TRAIL resistance in a large panel of melanoma cell lines. Expression of IK1 was also found in mitochondria, and its inhibition resulted in mitochondrial membrane hyperpolarization and an early activation of Bax. The combination of TRAM-34 and TRAIL resulted in massive release of mitochondrial factors, cytochrome c, AIF and SMAC/DIABLO. Bax knockdown and Bcl-2 overexpression abolished apoptosis. Overexpression of XIAP diminished apoptosis by two-fold, and SMAC knockdown almost completely abolished apoptosis. These data uncover the existence of a rheostat in melanoma cells, consisting of inhibitor of apoptosis proteins and SMAC, which regulates TRAIL sensitivity. Thus, a new strategy is described based on mitochondrial membrane channels, which correspond to Bax activation. As both TRAIL and IK1 inhibitors had shown only minor side effects in clinical trials, a clinical application of this combination is conceivable.     

Regulation of cell proliferation by intermediate-conductance Ca2+-activated potassium and volume-sensitive chloride channels in mouse mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine; however, their cellular physiology is not fully understood. The present study aimed at exploring the potential roles of the two dominant functional ion channels, intermediate-conductance Ca(2+)-activated potassium (IK(Ca)) and volume-sensitive chloride (I(Cl.vol)) channels, in regulating proliferation of mouse MSCs. We found that inhibition of IK(Ca) with clotrimazole and I(Cl.vol) with 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) reduced cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific short interference (si)RNAs significantly reduced IK(Ca) or I(Cl.vol) density and channel protein and produced a remarkable suppression of cell proliferation (by 24.4 +/- 9.6% and 29.5 +/- 7.2%, respectively, P < 0.05 vs. controls). Flow cytometry analysis showed that mouse MSCs retained at G(0)/G(1) phase (control: 51.65 +/- 3.43%) by inhibiting IK(Ca) or I(Cl.vol) using clotrimazole (2 microM: 64.45 +/- 2.20%, P < 0.05) or NPPB (200 microM: 82.89 +/- 2.49%, P < 0.05) or the specific siRNAs, meanwhile distribution of cells in S phase was decreased. Western blot analysis revealed a reduced expression of the cell cycle regulatory proteins cyclin D1 and cyclin E. Collectively, our results have demonstrated that IK(Ca) and I(Cl.vol) channels regulate cell cycle progression and proliferation of mouse MSCs by modulating cyclin D1 and cyclin E expression.