The Intermediate Conductance Calcium-activated Potassium Channel KCa3.1 Regulates Vascular Smooth Muscle Cell Proliferation via Controlling Calcium-dependent Signaling*

Background: The mechanism by which KCa3.1 regulates cell proliferation remains elusive. Results: KCa3.1 regulates the expression of transcription factors and cyclins by controlling intracellular calcium levels in activated vascular smooth muscle cells (VSMCs). Conclusion: KCa3.1 is an important regulator of the calcium-dependent proliferation machinery in VSMCs. Significance: KCa3.1 modulation constitutes a therapeutic target for cell proliferative diseases such as atherosclerosis. The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca2+ concentration ([Ca2+]i) and was enhanced by elevating [Ca2+]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca2+]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca2+]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF β-receptors and inhibiting the rise in [Ca2+]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca2+-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis.

The intermediate conductance calcium-activated potassium channel KCa3.1 (also known as KCNN4 and IKCa), a member of the calcium-activated potassium channel (KCa) 4 family, tightly binds the Ca 2ϩ sensor calmodulin near its C-terminal domain. KCa3.1 is opened by a small rise in free cytosolic Ca 2ϩ ([Ca 2ϩ ] i ) due to Ca 2ϩ -calmodulin-mediated cross-linking in the subunits of the channel tetramer (1). Channel activation induces membrane hyperpolarization, which promotes Ca 2ϩ influx. KCa3.1 is highly expressed in a variety of nonexcitable and proliferating cells (2), and an increase in KCa3.1 expression has been associated with cancer development, immune disorders, and vascular inflammation. In particular, this channel plays a critical role in the proliferation of smooth muscle cells (3,4), endothelial cells (5), lymphocytes including B-and T-cells (6,7), fibroblasts (8), stem cells (9), and several cancer cells (10).
Vascular smooth muscle cell (VSMC) proliferation is a crucial event in the development of vascular diseases such as atherosclerosis and restenosis (11,12). VSMCs proliferate, migrate, and invade the intima in response to growth factors and vascular injury, resulting in atherosclerotic fibrous cap formation and intimal hyperplasia following angioplasty and stent placement. Electrophysiological properties of VSMCs dramat-ically change as they proliferate (13). In the contractile form of VSMCs, Ca 2ϩ influx through voltage-dependent Ca 2ϩ channels causes VSMC contraction and co-activation of large conductance KCa channels (BK), which in turn induces Ca 2ϩ channel closure through repolarization. In contrast, in proliferating VSMCs, this mechanism is down-regulated, and Ca 2ϩ influx through Ca 2ϩ release-activated Ca 2ϩ channels is maintained by membrane hyperpolarization induced by KCa3.1 activation. We recently found that vascular remodeling following myocardial infarction (14) and chronic inhibition of nitric oxide synthesis (15) is associated with increased KCa3.1 expression in rat hearts, suggesting a pathophysiological role for this channel. Consistently, KCa3.1 expression and activity are increased in VSMCs activated by mitogens, in intimal VSMCs of restenotic lesions following vascular injury, and in atherosclerotic plaques in humans, swine, rats, and mice (3,11,12,16). Importantly, pharmacological blockade, siRNA knockdown, or genetic deficiency of KCa3.1 suppresses VSMC activation (including proliferation, migration, and excessive oxidant production) and attenuates the development of restenosis and atherosclerosis in swine, rats, and mice (3,12,17,18). Activation of the transcription factor AP-1 is associated with KCa3.1 induction in T cells (7). In VSMCs, an elevation of AP-1 activity is linked with proliferation (19,20), implying that this transcription factor may play a role in KCa3.1 induction in VSMCs. In addition, the repressor element 1-silencing transcription factor (REST) represses KCa3.1 gene expression (21), suggesting that AP-1 and REST may operate in a coordinated manner for KCa3.1 induction in VSMCs. Although a rise in [Ca 2ϩ ] i plays a crucial role in the regulation of cell proliferation via controlling intracellular signaling pathways (13,22,23), the mechanism by which KCa3.1 regulates cell proliferative processes remains unknown.
We hypothesized that KCa3.1 blockade or knockdown would suppress VSMC proliferation by inhibiting the mitogen-induced rise in [Ca 2ϩ ] i and subsequent mitogenic signaling pathways, whereas KCa3.1 stimulation or overexpression would confer the opposite effects. To test this hypothesis, we examined the effect of pharmacological KCa3.1 blockade or activation, gene silencing, or overexpression on platelet-derived growth factor-BB (PDGF)-induced proliferation of VSMCs, focusing on changes in [Ca 2ϩ ] i , activation of mitogenic signaling pathways, cell cycle progression, morphology, and phenotypic characteristics in human coronary artery VSMCs (HCSMCs).

EXPERIMENTAL PROCEDURES
Cell Culture-HCSMCs (Cell Applications) were grown as reported previously (3). All experiments were performed between passages 5 and 7. Cells were seeded and cultured up to 70% confluence in smooth muscle growth medium (Cell Applications). Before each assay, cells were serum-starved and synchronized for 48 h in smooth muscle basal medium. HCSMCs in a quiescent state were stimulated for 1-48 h with PDGF (20 ng/ml; R&D Systems). None of the treatments performed in this study altered the viability of VSMCs, as judged by trypan blue exclusion (data not shown).
RNA Extraction, Reverse Transcription, and Quantitative PCR-Real-time PCR (iCycler, Bio-Rad or 7900 HT real-time PCR system, Applied Biosystems) was performed using iQ SYBR Green supermix to quantify transcript levels for KCa3.1 and GAPDH as described (3). Primers were designed using Beacon Designer software 3.0 (PREMIER Biosoft International).
Cell Proliferation and Migration Assays-Cell proliferation (ELISA kit, Roche Applied Science) and migration were examined as we reported previously (3). VSMC migration was stimulated with PDGF for 8 h in Transwell plates (Corning).
Cell Cycle Analysis-As reported previously (3), HCSMCs were stimulated with 10% FBS for 24 h. After fixation, cells were treated with ribonuclease A (250 g/10 6 cells) for 30 min at 37°C and stained with propidium iodide (50 g/ml) for 30 min at 4°C. The ratio of cells in each cell cycle phase was determined by flow cytometry (FACSCalibur: BD Biosciences) using CellQuest software (BD Biosciences).
Intracellular Ca 2ϩ Measurement-To measure [Ca 2ϩ ] i in VSMCs after 48 h of treatment (24), cells were incubated with Fluo-4 AM (10 M; Invitrogen) for 25 min at room temperature. Fluorescence images were captured and analyzed using an inverted epifluorescence microscope (Nikon TE200) with a 40ϫ Plan Fluor objective, a high speed wavelength switcher (Lambda DG-4 from Sutter Instrument), a PC-controlled digital CCD camera (Hamamatsu C4742-95), and MetaMorph software (Universal Imaging). Fluorescence was measured at 488 nm, and recording emission was measured at 523 nm. Images were analyzed with MetaMorph software.
Patch Clamping-Serum-starved VSMCs or cells treated for 48 h with PDGF or with PDGF in the presence of TRAM-34 (a specific blocker of KCa3.1 (3,25)), EBIO (an activator of KCa3.1 and small conductance KCa2 channels (26)), or SKA-31 or NS309 (more potent and specific KCa3.1 activators (27,28)) were patch-clamped in the whole-cell mode of the patch clamp technique using an EPC-10 amplifier. KCa3.1 currents were elicited by dialysis with an aspartate-based pipette solution containing 3 M free Ca 2ϩ and voltage ramps from Ϫ120 to 40 mV of 200-ms duration applied every 10 s. Whole-cell KCa3.1 conductances were calculated from the slope at Ϫ80 mV where the KCa3.1 currents are not "contaminated" by inwardly rectifying potassium channel, voltage-gated potassium channel (Kv), or BK currents. The KCa3.1 whole-cell conductance was then divided by the KCa3.1 single channel conductance (11 picosiemens) to determine the KCa3.1 channel number per cell.
Determination of Cell Morphology-Cell morphology was analyzed as reported previously (29). After 48 h of treatment, phase-contrast images of ϳ20 randomly chosen fields per condition and per experiment were taken. The following morphological parameters were calculated from the cell boundary. 1) Cell length was determined along the principal axis of traction, which is a unique axis and presumably coincides with the principal actin bundles. 2) Cell width was measured in the direction perpendicular to the principal axis of traction. The maximum cell width was taken, ignoring thin cell protrusions. 3) The shape index was defined as the ratio of the cell length and width.
KCa3.1 Overexpression-For pharmacological induction, HCSMCs were treated with a combination of phorbol-12-myristate-13-acetate (PMA, 40 nM, a specific activator of protein kinase C; Calbiochem) and cyclosporin A (CsA, 100 nM, an inhibitor of calcineurin; Sigma-Aldrich) in smooth muscle basal medium for 48 h (7). For viral induction, replication-defective lentiviral vectors pseudotyped with vesicular stomatitis virus G-protein were produced as described previously (30). Positive colonies expressing eGFP were identified by fluorescent microscopy 3 days after the final transduction step. Using this approach, 50 -75% of HCSMCs were eGFP-positive.
Statistics-All data are expressed as mean Ϯ S.E. Student's t test and analysis of variance (for one-way and nonparametric tests) were performed using SigmaStat version 3 (SPSS Inc.) (3). Computations were followed by a Bonferroni's corrected t test when significant differences were noted. Statistical significance was defined as a value of p Ͻ 0.05.
Paradoxical Inhibition of PDGF-induced HCSMC Proliferation by KCa3.1 Stimulation-To test the hypothesis that full activation of KCa3.1 with pharmacological activators would enhance VSMC proliferation during PDGF exposure by augmenting the rise in [Ca 2ϩ ] i , EBIO (an activator of KCa3.1 and small conductance KCa2 channels (26)) was applied at 100 or 300 M during PDGF-induced proliferation of HCSMCs. These cells predominantly express KCa3.1 but not small conductance KCa2 channels (3). PDGF-induced increase in DNA synthesis was unexpectedly inhibited by co-treatment with EBIO in a dose-dependent manner, whereas EBIO alone had no significant effect (Fig. 3A). The antiproliferative effect of KCa3.1 stimulation with EBIO was also confirmed using a cell count assay (data not shown). PDGF-induced chemotaxis of HCSMCs was also inhibited by 300 M EBIO (fold increase: PDGFϩEBIO 7.5 Ϯ 3.0, p Ͻ 0.05 versus PDGF 17.4 Ϯ 4.0, n ϭ 5), and EBIO alone had no effect (EBIO 1.2 Ϯ 0.1). Fluorescence microscopy using Fluo-4 revealed that co-treatment with EBIO (300 M) reduced the PDGF-induced rise in [Ca 2ϩ ] i in HCSMCs (Fig.  3B). To confirm the antiproliferative effect of KCa3.1 stimulation, two more potent and specific KCa3.1 activators, SKA-31 (27) and NS309 (28), were also tested. SKA-31 inhibited PDGFinduced proliferation at 0.5 M, whereas SKA-16, an inactive derivative of SKA-31, had no effect (Fig. 3C). NS309 also exhibited an antiproliferative effect at 10 nM. Both activators had no effect in the absence of PDGF (data not shown).
To clarify the mechanisms responsible for this paradoxical inhibition, we first analyzed the expression level of KCa3.1 in HCSMCs treated with PDGF in the presence of TRAM-34 or the KCa3.1 activators. As we reported previously (3) KCa3.1 Regulates Cell Cycle Progression-We next studied the effect of TRAM-34 or EBIO treatment on the activation of CREB and on the expression of c-Fos, two factors playing an important role in PDGF-induced VSMC proliferation (32,35).
Western blot experiments showed that PDGF induced CREB phosphorylation at Ser-133 and c-Fos up-regulation in HCSMCs (Fig. 4A), which were inhibited by both TRAM-34 and EBIO (100 nM and 300 M, respectively). Consistent with

KCa3.1 and Vascular Smooth Muscle Cell Proliferation
the fact that the CREB/c-Fos pathway is associated with cell cycle progression via regulating cyclin expression (35), PDGF treatment increased the expression of cyclins A, B1, D1, and E in HCSMCs (Fig. 4B). TRAM-34 reduced the expression of cyclins A and B1, and EBIO suppressed all tested cyclins. Flow cytometry analysis of the cellular DNA content showed that 10% FBS evoked cell cycle progression with fewer G 0 /G 1 and more S and G 2 /M cells (Fig. 4C), which was reduced by TRAM-34 (100 nM) at the transitions from G 0 /G 1 to S and from S to G 2 /M phases, whereas EBIO (300 M) more remarkably inhibited cell cycle progression, especially at the transition from G 0 /G 1 to S phase. siRNA knockdown of KCa3.1 also inhibited cell cycle progression in a manner similar to EBIO (data not shown). These data indicate that KCa3.1 plays a crucial role in the regulation of cell cycle signaling pathways during VSMC proliferation.

JOURNAL OF BIOLOGICAL CHEMISTRY 15847
VSMC phenotype to one that is resistant to PDGF-induced proliferation by disrupting the axis of PDGF ␤-receptors and its Ca 2ϩ regulatory mechanism.
KCa3.1 Overexpression Initiates and Enhances VSMC Proliferation-Two methods of pharmacological and genetic manipulations were used to increase KCa3.1 expression in quiescent VSMCs. As reported in T cells (7), treatment with PMA and CsA for 48 h increased KCa3.1 mRNA and protein expression with little de novo DNA synthesis (PMAϩCsA treatment 1.0 Ϯ 0.2-fold of control, p ϭ not significant versus control, n ϭ 3) in HCSMCs, which can be compared with KCa3.1 expression 48 h after exposure to PDGF (Fig. 6A). Following removal of PMAϩCsA, these cells displayed a modest but statistically significant proliferative activity in the absence of any mitogen as compared with quiescent cells (Fig. 6B). PDGF-induced proliferation was also enhanced in PMAϩCsA-pretreated, KCa3.1overexpressing cells 24 h after exposure to PDGF as compared with control cells. A lentiviral vector system was subsequently utilized to genetically modify HCSMCs with either eGFP-tagged hKCa3.1 (KCa3.1-tg cells) or eGFP-tagged luciferase (Luc-tg cells) as a control. As confirmed by the expression of eGFP, KCa3.1-tg cells showed a high level of KCa3.1 mRNA and protein expression as compared with Luc-tg cells (Fig. 7A). BrdU incorporation assay performed in the absence of PDGF revealed that the activity of de novo DNA synthesis in KCa3.1-tg cells was higher than those in nontransduced and Luc-tg cells (Fig. 7B). PDGFinduced proliferation was also enhanced in KCa3.1-tg cells (Fig.  7B). The levels of phosphorylated CREB, c-Fos, and NOR-1 were higher in KCa3.1-tg cells than in Luc-tg cells before, and 1 h after, exposure to PDGF (Fig. 7C). These data confirm that KCa3.1 regulates PDGF-dependent signaling pathways of VSMC proliferation.

DISCUSSION
The major novel findings of this study are three-fold. First, both pharmacological blockade and gene knockdown of KCa3.1 inhibit HCSMC proliferation, whereas KCa3.1 overexpression has the opposite effect. Second, pharmacological KCa3.1 stimulation unexpectedly but markedly attenuates HCSMC proliferation by diminishing the expression of KCa3.1 and PDGF ␤-receptors and by inhibiting further dedifferentiation and cell cycle progression. Third, PDGF-induced HCSMC proliferation is associated with KCa3.1-dependent regulation of the rise in [Ca 2ϩ ] i and the subsequent expression of transcription factors and cyclins that orchestrate cell cycle progression. Taken together, these findings suggest that KCa3.1 plays a key role in the regulation of VSMC proliferation.
Ca 2ϩ is a major second messenger for a variety of cell activation processes. PDGF binds to and activates its specific tyrosine kinase receptors, leading to a sustained increase in [Ca 2ϩ ] i in VSMCs (31). Here, PDGF-induced HCSMC proliferation was suppressed by chelating [Ca 2ϩ ] i with BAPTA and augmented by a forced rise in [Ca 2ϩ ] i with A23187. These results are consistent with the effects of BAPTA and A23187 on PDGF-induced VSMC chemotaxis (31), indicating an essential role for [Ca 2ϩ ] i in the regulation of PDGF-induced VSMC activation. It is widely accepted that in a variety of nonexcitable cells, KCa3.1 is activated by a small rise in [Ca 2ϩ ] i (ϳ100 nM) following Ca 2ϩ release from intracellular stores and subsequent Ca 2ϩ influx. The resultant K ϩ efflux causes membrane hyperpolarization that maintains Ca 2ϩ entry by increasing an electrical gradient, thereby playing a role in the activation of Ca 2ϩ -dependent signal pathways (38). In VSMCs, an increase in [Ca 2ϩ ] i activates specific signaling pathways. CREB, a mitogen-induced transcriptional factor, is phosphorylated in response to a rise in [Ca 2ϩ ] i and subsequently induces mitogenic immediate early genes such as c-fos and NOR-1 (32)(33)(34). In the present study, blockade or siRNA-mediated targeting of KCa3.1 suppressed PDGF-induced HCSMC proliferation, concomitantly with an inhibition of the rise in [Ca 2ϩ ] i , CREB phosphorylation, and immediate early gene expression, and these effects were abolished by a forced rise in [Ca 2ϩ ] i with A23187. In contrast, KCa3.1 overexpression induced and enhanced VSMC proliferation by activating these signaling pathways. KCa3.1 is associ-ated with VSMC proliferation to a variety of mitogens in different species (3,12,39). Taken together, these data indicate that VSMC proliferation is associated with KCa3.1-dependent regulation of Ca 2ϩ -dependent signaling pathways.
EBIO at concentrations relatively specific to KCa3.1 (26) in HCSMCs (which predominantly express KCa3.1 but not small conductance KCa2 channels (3)) also suppressed PDGF-induced rise in [Ca 2ϩ ] i , proliferation, and migration, accompanied by lower expression and activity of KCa3.1 and PDGF ␤-receptors. SKA-31 and NS309, two chemically distinct and much more potent KCa3.1 activators, had similar effects. The following mechanisms may explain the inhibitory effects of KCa3.1 activators. 1) Because membrane potential is crucial for cell proliferation (40,41), excessive membrane hyperpolarization due to full activation of KCa3.1 (3) will stop proliferation in a nonspecific manner. 2) Upon PDGF stimulation, KCa3.1 activators stimulate a small number of KCa3.1 channels remaining on the cell membrane (3). The resultant over-hyperpolarization causes an excess of Ca 2ϩ entry that favors cell differentiation rather than proliferation (43). 3) Consistent with a study in HaCaT keratinocyte and C6 glioma cell lines where EBIO also inhibited proliferation (42), an incisive general mechanism of negative feedback abolishes KCa3.1 expression, leading to lowered [Ca 2ϩ ] i and thereby attenuated proliferation during the prolonged stimulation. It is unlikely that these activators nonspecifically evoke these effects, independently of KCa3.1 stimulation, because the morphological changes induced by EBIO were suppressed by TRAM-34; SKA-16, an inactive analog of SKA-31, had no effects; and KCa3.1-deficient VSMCs exhibit an almost complete loss of proliferative response to PDGF (3). As compared with TRAM-34, EBIO treatment resulted in a stronger inhibition of PDGF-induced signaling pathways and cell cycle progression, with a lesser induction of cyclins. This effect could be explained by the fact that KCa3.1 stimulation abolished the expression of PDGF ␤-receptors, whereas TRAM-34 had no additive effect to the PDGF-induced downregulation of this receptor (36). Interestingly, the KCa3.1 activators were also more effective at reducing PDGF-induced increases in KCa3.1 expression. In addition, a forced rise in [Ca 2ϩ ] i with A23187 restored the proliferative response in KCa3.1 blocker-treated VSMCs (where [Ca 2ϩ ] i levels were lowered but the expression of PDGF ␤-receptors was unaltered) but not in KCa3.1 activator-treated cells (where both [Ca 2ϩ ] i levels and PDGF ␤-receptor expression were reduced). Therefore, the abolishment of PDGF ␤-receptor expression is an additional mechanism for diminished responsiveness to PDGF in KCa3.1 activator-treated VSMCs. Indeed, we show here that treatment with KCa3.1 activators prevents PDGF-induced upregulation of l-caldesmon and down-regulation of calponin-1. Because the mechanisms of PDGF-induced KCa3.1 gene activation in VSMCs remain to be determined, the precise mecha-nism by which KCa3.1 activators abolish KCa3.1 gene expression needs further investigation.
Cyclins bound to specific cyclin-dependent kinases control cell cycle progression; in particular, cyclin D1 mainly controls G 1 phase, cyclins A and E control S phase, whereas cyclins A and B1 control mitotic phase (44). In addition, a rise in [Ca 2ϩ ] i , phosphorylated CREB, and immediate early genes play important roles in the expression and activation of cyclins, in the induction of resting cell (G 0 ) reentry into the cell cycle, in DNA synthesis at G 1 /S transition, and in mitosis at G 2 /M transition in a cooperative fashion (45)(46)(47). In this study, TRAM-34 inhibited PDGF-induced expression of cyclins A and B1, but not D1 and E, along with a reduction in the rise in [Ca 2ϩ ] i , CREB phosphorylation, and c-Fos and NOR-1 expression. This effect is consistent with the finding that TRAM-34 inhibited FBS-induced cell cycle progression at transitions from G 1 to S and G 2 to M phases. In contrast, EBIO more effectively inhibited FBS-induced cell cycle progression at the transition from G 1 to S phase, concomitantly with a stronger inhibition in PDGF-induced expression of c-Fos and cyclins A, B1, D1, and E, indicating that KCa3.1 stimulation could prevent the reentry of resting cells (G 0 ) into the cell cycle.
PDGF induces VSMC elongation and thinning through the migratory machinery. Our previous observation that blockade of KCa3.1 inhibits PDGF-induced VSMC migration (3) is consistent with the present study showing that TRAM-34 inhibits PDGF-induced cell elongation and thinning, whereas KCa3.1 stimulation enhances these changes in a TRAM-34-sensitive manner. Thus, KCa3.1 may play a crucial role in the regulation of protrusive activity. Other possible mechanisms include the contribution of KCa3.1 to cell volume regulation that modulates morphological changes and the proliferative response (48,49). Furthermore, in contrast to the effects of TRAM-34, a forced rise in [Ca 2ϩ ] i with A23187 failed to suppress the inhibitory effect of EBIO on proliferation. KCa3.1 stimulation decreased l-caldesmon and PDGF ␤-receptor expression, VSMC dedifferentiation markers, and increased calponin-1 expression, a differentiation marker. These effects were accompanied by a strong suppression of KCa3.1 expression, the appearance of a spindle-like shape, and the suppression of signaling pathways and cyclin expression. Thus, the effects of KCa3.1 stimulation cannot be explained only by their direct effects on channel function, implying a possible link between KCa3.1 and VSMC differentiation/dedifferentiation genes. KCa3.1 overexpression induced VSMC proliferation in the absence of PDGF via signaling pathways similar to those activated by this growth factor. Moreover, KCa3.1 overexpression also enhanced the response to PDGF. Similar enhanced cell proliferation has been reported for the ether-a-go-go K ϩ channel (Kv10.1), which also regulates Ca 2ϩ influx and is expressed in up to 70% of human cancers (50). Possible mechanisms include: 1) increased channel expression may cause membrane hyperpolarization and Ca 2ϩ influx through nonspecific cation channels, leading to CREB-dependent activation of mitogenic genes (33), and 2) KCa3.1 may be increased in intracellular organelles such as mitochondria, and may maintain the driving force for the store Ca 2ϩ efflux with counter-influx of K ϩ (51).
In summary, KCa3.1 blockade or knockdown reduces PDGFinduced VSMC proliferation via inhibiting Ca 2ϩ -dependent signaling pathways and cell cycle progression, whereas KCa3.1 overexpression has the opposite effects. Pharmacological KCa3.1 stimulation suppresses these responses more markedly via abolishing KCa3.1 and PDGF ␤-receptor expression. Therefore, targeting KCa3.1 with specific activators, in addition to blockers and gene therapy, may constitute a new therapeutic approach for the prevention of diseases with increased cell proliferative activity such as atherosclerosis.