7Kv channels constitute a large and ubiquitous family of membrane proteins present in both excitable and nonexcitable cells. In nonexcitable cells, their function as feedback regulators of resting V M has been proposed to participate in many cellular functions ranging from secretion to cell migration, proliferation, and apoptotic death. Kv channel genes can give rise to an even larger number of functional Kv currents, via heteromultimerization, association with accessory subunits, Kv1 .3 Channels Can Modulate Cell Proliferation during Phenotypic switch by an ion-Flux independent MechanismPilar Cidad,* Laura Jiménez-Pérez,* Daniel García-Arribas, Eduardo Miguel-Velado, Sendoa Tajada, Christian Ruiz-McDavitt, José R. López-López, † M. Teresa Pérez-García † Objective-Phenotypic modulation of vascular smooth muscle cells has been associated with a decreased expression of all voltage-dependent potassium channel (Kv)1 channel encoding genes but Kcna3 (which encodes Kv1.3 channels). In fact, upregulation of Kv1.3 currents seems to be important to modulate proliferation of mice femoral vascular smooth muscle cells in culture. This study was designed to explore if these changes in Kv1 expression pattern constituted a landmark of phenotypic modulation across vascular beds and to investigate the mechanisms involved in the proproliferative function of Kv1.3 channels. Methods and Results-Changes in Kv1.3 and Kv1.5 channel expression were reproduced in mesenteric and aortic vascular smooth muscle cells, and their correlate with protein expression was electrophysiologicaly confirmed using selective blockers. Heterologous expression of Kv1.3 and Kv1.5 channels in HEK cells has opposite effects on the proliferation rate. The proproliferative effect of Kv1.3 channels was reproduced by "poreless" mutants but disappeared when voltagedependence of gating was suppressed. Conclusion-These

Essential hypertension involves a gradual and sustained increase in total peripheral resistance, reflecting an increased vascular tone. This change associates with a depolarization of vascular myocytes, and relies on a change in the expression profile of voltage-dependent ion channels (mainly Ca 2+ and K + channels) that promotes arterial contraction. However, changes in expression and/or modulation of voltage-dependent K + channels (Kv channels) are poorly defined, due to their large molecular diversity and their vascular bed-specific expression. Here we endeavor to characterize the molecular and functional expression of Kv channels in vascular smooth muscle cells (VSMCs) and their regulation in essential hypertension, by using VSMCs from resistance (mesenteric) or conduit (aortic) arteries obtained from a hypertensive inbred mice strain, BPH, and the corresponding normotensive strain, BPN. Real-time PCR reveals a differential distribution of Kv channel subunits in the different vascular beds as well as arterial bed-specific changes under hypertension. In mesenteric arteries, the most conspicuous change was the de novo expression of Kv6.3 (Kcng3) mRNA in hypertensive animals. The functional relevance of this change was studied by using patch-clamp techniques. VSMCs from BPH arteries were more depolarized than BPN ones, and showed significantly larger capacitance values. Moreover, Kv current density in BPH VSMCs is decreased mainly due to the diminished contribution of the Kv2 component. The kinetic and pharmacological profile of Kv2 currents suggests that the expression of Kv6.3 could contribute to the natural development of hypertension.

IDE is not a rate-limiting regulator of plasma insulin levels in vivo.

Objective-Vascular smooth muscle cells (VSMCs) contribute significantly to occlusive vascular diseases by virtue of their ability to switch to a noncontractile, migratory, and proliferating phenotype. Although the participation of ion channels in this phenotypic modulation (PM) has been described previously, changes in their expression are poorly defined because of their large molecular diversity. We obtained a global portrait of ion channel expression in contractile versus proliferating mouse femoral artery VSMCs, and explored the functional contribution to the PM of the most relevant changes that we observed. Methods and Results-High-throughput real-time polymerase chain reaction of 87 ion channel genes was performed in 2 experimental paradigms: an in vivo model of endoluminal lesion and an in vitro model of cultured VSMCs obtained from explants. mRNA expression changes showed a good correlation between the 2 proliferative models, with only 2 genes, Kv1.3 and Kv␤2, increasing their expression on proliferation. The functional characterization demonstrates that Kv1.3 currents increased in proliferating VSMC and that their selective blockade inhibits migration and proliferation. Key Words: gene expression Ⅲ ion channels Ⅲ restenosis Ⅲ vascular biology Ⅲ vascular muscle Ⅲ Kv1.3 channels Ⅲ vascular remodeling V ascular smooth muscle cells (VSMCs) are differentiated cells that regulate vessel diameter and determine tissue perfusion. However, they can exhibit a variety of functionally dissimilar phenotypes. In response to local cues, VSMCs experience a phenotypic modulation (PM), with profound and reversible changes leading to proliferation, migration, and secretion of extracellular matrix components. 1 This plasticity is essential for injury repair, but it also contributes to the development and progression of vascular disease in response to abnormal environmental signals. It is becoming evident that contractile and proliferative phenotypes represent extreme cases of a spectrum of phenotypes that may coexist as the result of a developmentally regulated genetic program constantly modulated by environmental cues. This explains both a relatively stable expression of certain transcriptional programs in different VSMCs and a marked plasticity of these cells, including the ability to respond with different genetic programs to readjust cellular activity to mechanical and hormonal factors. [1][2][3] See accompanying article on page 1073 Conclusion-TheseThe switch in ion transport mechanisms associated with PM is getting increasing amounts of attention. Coordinate changes in ion channels are an integral component of VSMC plasticity, as they can redirect biochemical activity toward new functional responses. 4,5 Moreover, both contractile and proliferative signals require specific changes in intracellular [Ca 2ϩ ] and membrane potential that are determined by the ion channels expressed in VSMCs. Remodeling of several ion channels has shown to be functionally important for the PM of VSMCs in several preparations. [5][6][7][8]...

Kv1.3 channels are involved in the switch to proliferation of normally quiescent cells, being implicated in the control of cell cycle in many different cell types and in many different ways. They modulate membrane potential controlling K fluxes, sense changes in potential, and interact with many signaling molecules through their intracellular domains. From a mechanistic point of view, we can describe the role of Kv1.3 channels in proliferation with at least three different models. In the "membrane potential model," membrane hyperpolarization resulting from Kv1.3 activation provides the driving force for Ca influx required to activate Ca-dependent transcription. This model explains most of the data obtained from several cells from the immune system. In the "voltage sensor model," Kv1.3 channels serve mainly as sensors that transduce electrical signals into biochemical cascades, independently of their effect on membrane potential. Kv1.3-dependent proliferation of vascular smooth muscle cells (VSMCs) could fit this model. Finally, in the "channelosome balance model," the master switch determining proliferation may be related to the control of the Kv1.3 to Kv1.5 ratio, as described in glial cells and also in VSMCs. Since the three mechanisms cannot function independently, these models are obviously not exclusive. Nevertheless, they could be exploited differentially in different cells and tissues. This large functional flexibility of Kv1.3 channels surely gives a new perspective on their functions beyond their elementary role as ion channels, although a conclusive picture of the mechanisms involved in Kv1.3 signaling to proliferation is yet to be reached.

Abstract-Vascular smooth muscle cells (VSMCs) perform diverse functions that can be classified into contractile and synthetic (or proliferating). All of these functions can be fulfilled by the same cell because of its capacity of phenotypic modulation in response to environmental changes. The resting membrane potential is a key determinant for both contractile and proliferating functions. Here, we have explored the expression of voltage-dependent K ϩ (Kv) channels in contractile (freshly dissociated) and proliferating (cultured) VSMCs obtained from human uterine arteries to establish their contribution to the functional properties of the cells and their possible participation in the phenotypic switch. We have studied the expression pattern (both at the mRNA and at the protein level) of Kv␣ subunits in both preparations as well as their functional contribution to the K ϩ currents of VSMCs. Our results indicate that phenotypic remodeling associates with a change in the expression and distribution of Kv channels. Whereas Kv currents in contractile VSMCs are mainly performed by Kv1 channels, Kv3.4 is the principal contributor to K ϩ currents in cultured VSMCs. Furthermore, selective blockade of Kv3.4 channels resulted in a reduced proliferation rate, suggesting a link between Kv channels expression and phenotypic remodeling. T he vascular smooth muscle cells (VSMCs) of mature animals are highly specialized cells whose main function is contraction. Although during vasculogenesis, the principal role of VSMCs is proliferation and synthesis of the matrix components of the vessel wall, differentiated VSMCs proliferate at an extremely low rate and express a unique repertoire of contractile proteins, ion channels, and signaling molecules required for contraction. However, VSMCs can undergo relatively rapid and reversible phenotypic changes in response to local environmental conditions. Accelerated proliferation of VSMCs is known to play a key role in atherosclerotic plaque formation and postangioplasty restenosis and is a common feature in hypertensive arteries. 1 VSMCs express a large repertoire of ion channels and membrane receptors that vary widely among different vascular beds and are key determinants of the electrical and contractile responses of the cells. [2][3][4][5] Although release of intracellular Ca 2ϩ is necessary for effective contraction, Ca 2ϩ influx through voltage-activated Ca 2ϩ (Cav) channels is responsible for initiating contraction, making resting membrane potential (E m ) a primary determinant of vascular smooth muscle tone. 4 Membrane depolarization opens Cav and raises [Ca 2ϩ ] i , and cytosolic-free Ca 2ϩ serves, in turn, as a critical signal transduction element in a variety of cell functions, such as contraction, migration, proliferation, and gene expression. 6 -8 There is increasing evidence showing that K ϩ channels may have an important role in dedifferentiation and proliferation of VSMCs. Modulating E m , K ϩ channels can affect not only Ca 2ϩ influx, a well-established factor influen...

Changes in voltage-dependent potassium channels (Kv channels) associate to proliferation in many cell types, including transfected HEK293 cells. In this system Kv1.5 overexpression decreases proliferation, whereas Kv1.3 expression increases it independently of K ؉ fluxes. To identify Kv1.3 domains involved in a proliferation-associated signaling mechanism(s), we constructed chimeric Kv1.3-Kv1.5 channels and point-mutant Kv1.3 channels, which were expressed as GFP-or cherry-fusion proteins. We studied their trafficking and functional expression, combining immunocytochemical and electrophysiological methods, and their impact on cell proliferation. We found that the C terminus is necessary for Kv1.3-induced proliferation. We distinguished two residues (Tyr-447 and Ser-459) whose mutation to alanine abolished proliferation. The insertion into Kv1.5 of a sequence comprising these two residues increased proliferation rate. Moreover, Kv1.3 voltage-dependent transitions from closed to open conformation induced MEK-ERK1/2-dependent Tyr-447 phosphorylation. We conclude that the mechanisms for Kv1.3-induced proliferation involve the accessibility of key docking sites at the C terminus. For one of these sites (Tyr-447) we demonstrated the contribution of MEK/ERK-dependent phosphorylation, which is regulated by voltage-induced conformational changes.

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