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In heart cells, several distinct kinds of transient spatial patterns of cytoplasmic calcium ion concentration ([Ca2+]i) can be observed: (1) [Ca2+]i waves, in which regions of spontaneously increased [Ca2+]i propagate at high velocity (100 microns/s) through the cell; (2) Ca2+ 'sparks', which are spontaneous, non-propagating changes in [Ca2+]i that are localized in small (approximately 2 microns) subcellular regions; and (3) evoked [Ca2+]i transients that are elicited by electrical depolarization, in association with normal excitation-contraction (E-C) coupling. In confocal [Ca2+]i images, evoked [Ca2+]i transients appear to be nearly spatially uniform throughout the cell, except during their rising phase or during small depolarizations. In contrast to [Ca2+]i waves and spontaneous Ca2+ sparks, evoked [Ca2+]i transients are triggered by L-type Ca2+ channel current and they are 'controlled', in the sense that stopping the L-type Ca2+ current stops them. Despite their different characteristics, all three types of Ca2+ transient involve Ca(2+)-induced release of Ca2+ from the sarcoplasmic reticulum. Here, we address the question of how the autocatalytic process of Ca(2+)-induced Ca2+ release, which can easily be understood to underlie spontaneous regenerative ('uncontrolled'), propagating [Ca2+]i waves, might be 'harnessed', under other circumstances, to produce controlled changes in [Ca2+]i, as during normal excitation-contraction coupling, or changes in [Ca2+]i that do not propagate. We discuss our observations of Ca2+ waves, Ca2+ sparks and normal Ca2+ transients in heart cells and review our results on the 'gain' of Ca(2+)-induced Ca2+ release. We discuss a model involving Ca2+ microdomains beneath L-type Ca2+ channels, and clusters of Ca(2+)-activated Ca2+ release channels in the sarcoplasmic reticulum which may form the basis of the answer to this question.

El pdf del artículo es el manuscrito de autor., Hypoxic inhibition of K+ channels has been documented in many native chemoreceptor cells, and is crucial to initiate reflexes directed to improve tissue O2 supply. In the carotid body (CB) chemoreceptors, there is a general consensus regarding the facts that a decrease in PO2 leads to membrane depolarization, increase of Ca2+ entry trough voltage-dependent Ca2+ channels and Ca2+-dependent release of neurotransmitters. Central to this pathway is the modulation by hypoxia of K+ channels that triggers depolarization. However, the details of this process are still controversial, and even the molecular nature of these oxygen-sensitive K+ ((KO2)) channels in the CB is hotly debated. Clearly there are inter-species differences, and even in the same preparation more that one KO2 may be present. Here we recapitulate our present knowledge of the role of voltage dependent K+ channels as KO2 in the CB from different species, and their functional contribution to cell excitability in response to acute and chronic exposure to hypoxia. © 2007 Elsevier B.V. All rights reserved., This work was supported by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III grants R006/009 (Red Heracles) and PI041044 (JRLL), Ministerio de Educación y Ciencia grant BFU2004-05551 (MTPG) and Junta de Castilla y León grant VA011C05.

In this work we have combined biochemical and electrophysiological approaches to explore the modulation of rat ventricular transient outward K + current (Ito) by calmodulin kinase II (CaMKII). Intracellular application of CaMKII inhibitors KN93, calmidazolium, and autocamtide-2-related inhibitory peptide II (ARIP-II) accelerated the inactivation of Ito, even at low [Ca2+]. In the same conditions, CaMKII coimmunoprecipitated with Kv4.3 channels, suggesting that phosphorylation of Kv4.3 channels modulate inactivation of Ito. Because channels underlying Ito are heteromultimers of Kv4.2 and Kv4.3, we have explored the effect of CaMKII on human embryonic kidney (HEK) cells transfected with either of those Kvα-subunits. Whereas Kv4.3 inactivated faster upon inhibition of CaMKII, Kv4.2 inactivation was insensitive to CaMKII inhibitors. However, Kv4.2 inactivation became slower when high Ca2+ was used in the pipette or when intracellular [Ca2+] ([Ca2+]i) was transiently increased. This effect was inhibited by KN93, and Western blot analysis demonstrated Ca2+- dependent phosphorylation of Kv4.2 channels. On the contrary, CaMKII coimmunoprecipitated with Kv4.3 channels without a previous Ca2+ increase, and the association was inhibited by KN93. These results suggest that both channels underlying Ito are substrates of CaMKII, although with different sensitivities; Kv4.2 remain unphosphorylated unless [Ca 2+]i increases, whereas Kv4.3 are phosphorylated at rest. In addition to the functional impact that phosphorylation of Kv4 channels could cause on the shape of action potential, association of CaMKII with Kv4.3 provides a new role of Kv4.3 subunits as molecular scaffolds for concentrating CaMKII in the membrane, allowing Ca2+-dependent modulation by this enzyme of the associated Kv4.2 channels. Copyright © 2006 the American Physiological Society., This work was supported by Instituto de Salud Carlos III Grants G03/011 (Red Respira), G03/045 (Red Heracles), and PI041044 to J. R. López-López, Ministerio de Educación (MEC) Grant BFU2004-05551 to M. T. Pérez-García and MEC Grant SAF2005-00906 to O. Casis. O. Colinas is a fellow of the Spanish Ministerio de Ciencia y Tecnología, and M. Gallego and R. Setién are fellows of the University of Basque Country.

Producción Científica, 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+ (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 Kvα subunits in both preparations as well as their functional contribution to the K+ 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+ 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., Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grants R006/009. FS041139-0 and PI041044), Ministerio de Ciencia, Innovación y Universidades (grants BFU2004-05551 and BFU2007-61524), Junta de Castilla y León (grant GR242)

Carotid body (CB) chemoreceptors are the major contributors to the hyperventilatory responses to acute hypoxia. Low PO2 inhibits K channel activity of chemoreceptor cells, inducing a depolarization-mediated neurotransmitter release that elicits the ventilatory response (Gonzalez et al, 1994). Although O2-sensitive K+ currents were described in rabbit CB chemoreceptor cells several years ago (Lopez-Barneo et ai, 1988), the molecular identity of the underlying K channels remains unresolved.

The arterial carotid body (CB) chemoreceptors are highly vascularized sensory organs located in the proximity of the carotid artery bifurcation and formed by clusters of parenchymal cells. The cell clusters are penetrated by sensory fibers of the carotid sinus nerve (CSN) which form synapses with the parenchymal chemoreceptor cells. Functionally, the CBs are the origin of a regulatory loop devoted to restore O2 availability in situations of hypoxia (Richalet, 1997). To achieve this function chemoreceptor cells detect blood PO2, being activated by hypoxia. Hypoxia increases the rate of the release of neurotransmitters from the cells augmenting the action potential frequency in the CSN; this increased activity stimulates the brainstem regulators of respiration and hyperventilation and increased arterial blood PO2 ensue (Gonzalez et al., 1994).

The notion that intracellular Ca2+(Cai2+) stores play a significant role in the chemoreception process in chemoreceptor cells of the carotid body (CB) appears in the literature in a recurrent manner. However, the structural identity of the Ca2+stores and their real significance in the function of chemoreceptor cells are unknown. To assess the functional significance of Cai2+stores in chemoreceptor cells, we have monitored 1) the release of catecholamines (CA) from the cells using an in vitro preparation of intact rabbit CB and 2) the intracellular Ca2+concentration ([Ca2+]i) using isolated chemoreceptor cells; both parameters were measured in the absence or the presence of agents interfering with the storage of Ca2+. We found that threshold [Ca2+]ifor high extracellular K+(Ke+) to elicit a release response is ≈250 nM. Caffeine (10–40 mM), ryanodine (0.5 μM), thapsigargin (0.05–1 μM), and cyclopiazonic acid (10 μM) did not alter the basal or the stimulus (hypoxia, high Ke+)-induced release of CA. The same agents produced Cai2+transients of amplitude below secretory threshold; ryanodine (0.5 μM), thapsigargin (1 μM), and cyclopiazonic acid (10 μM) did not alter the magnitude or time course of the Cai2+responses elicited by high Ke+. Several potential activators of the phospholipase C system (bethanechol, ATP, and bradykinin), and thereby of inositol 1,4,5-trisphosphate receptors, produced minimal or no changes in [Ca2+]iand did not affect the basal release of CA. It is concluded that, in the rabbit CB chemoreceptor cells, Cai2+stores do not play a significant role in the instant-to-instant chemoreception process.

Almitrine is a drug used in the treatment of hypoxemic chronic lung diseases such as bronchitis and emphysema because it is a potent stimulant of the carotid bodies in human and different animal species that produces a long-lasting enhancement of alveolar ventilation, ameliorating arterial blood gases. However, the mechanism of action of almitrine remains unknown. We investigated the effect of almitrine on ionic currents of chemoreceptor cells isolated from the carotid body of rat and rabbits by using the whole-cell and inside-out configurations of the patch-clamp technique. Almitrine at concentrations up to 10 microM did not affect whole-cell voltage-dependent K+, Ca2+, or Na+ currents in rat or rabbit cells. However, this concentration of almitrine significantly inhibited the Ca2+-dependent component of K+ currents in rat chemoreceptor cells. This effect of almitrine on the Ca2+-dependent component of K+ currents was investigated further at the single-channel level in excised patches in the inside-out configuration. In this preparation, almitrine inhibited the activity of a high-conductance (152 +/- 13 pS), Ca2+-dependent K+ channel by decreasing its open probability. The IC50 value of the effect was 0. 22 microM. The inhibitory effect of almitrine on Ca2+-dependent K+ channels also was observed in GH3 cells. We conclude that almitrine inhibits selectively the Ca2+-dependent K+ channel and that in rat chemoreceptor cells, this inhibition could represent an important mechanism of action underlying the therapeutic actions of the drug.

1. The electrical properties of chemoreceptor cells from neonatal rat and adult rabbit carotid bodies (CBs) are strikingly different. These differences have been suggested to be developmental and/or species related. To distinguish between the two possibilities, the whole-cell configuration of the patch-clamp technique was used to characterize the ionic currents present in isolated chemoreceptor cells from adult rat CBs. Since hypoxia-induced inhibition of O2-sensitive K+ currents is considered a crucial step in O2 chemoreception, the effect of hypoxia on the adult rat chemoreceptor cell currents was also studied. 2. Outward currents were carried mainly by K+, and two different components could be distinguished: a Ca(2+)-dependent K+ current (IK(Ca)) sensitive to Cd2+ and charybdotoxin (CTX), and a Ca(2+)-insensitive, voltage-dependent K+ current (IK(V)). IK(V) showed a slow voltage-dependent activation (time constant (tau) of 87.4 ms at -20 mV and 8.8 ms at +60 mV) and a very slow inactivation, described by the sum of two exponentials (tau 1 = 684 +/- 150 ms and tau 2 = 4.96 +/- 0.76 s at + 30 mV), that was almost voltage insensitive. The kinetic and pharmacological properties of IK(V) are typical of a delayed rectifier K+ channel. 3. Voltage-dependent Ca2+ currents (ICa) were present in nineteen of twenty-seven cells. TTX-sensitive Na+ currents were also observed in about 10% of the cells. 4. Low PO2 (< 10 mmHg) reduced the whole outward current amplitude by 22.17 +/- 1.96% (n = 27) at +20 mV. This effect was absent in the presence of Cd2+. Since low PO2 did not affect ICa, we conclude that hypoxia selectively blocks IK(Ca). 5. The properties of the currents recorded in adult rat chemoreceptor cells, including the specific inhibition of IK(Ca) by hypoxia, are similar to those reported in neonatal rat CB cells, implying that the differences between rat and rabbit chemoreceptor cells are species related.

Producción Científica, The pívotal role of íntracellular free [Ca2+] fluctuatíons in the control of cellular functíons such as contraction and secretíon, íncludíng the release of neurotransmítters, was recognized many decades ago (see Rubín, 1982). More recently, the list of cellular functíons tríggered or modulated by the levels of Ca2+¡ has grown enormously. Addítional functíons regulated by [Ca2+)¡ include neuronal excítabílity, synaptic plastícíty, gene ex­ pressíon, cellular metabolísm, cell dívísíon and dífferentíatíon, and programmed cell dead (Míller, 1991; Clapham, 1995). Parallelíng the growth in this líst of Ca2+-controlled func­ tíons, a multíplicity of cellular mechanísms aimed at maintaining resting free [Ca2+)¡ in the range of l 00 nM for most cells has been described, allowing increases in Ca2+¡ levels that are specific in their magnitude, time course and spatial distributíon, accordíng to the cell function activated (Toescu, 1995). Since Ca2+ cannot be metabolized, cells regulate theír cytoplasmic levels of free Ca2+ through numerous bínding proteíns and influx and efflux mechanisms (Fíg 1). Ca2+ ínflux to cell cytoplasm from the extracellular milieu occurs vía voltage or receptor operated channels or vía yet ill-defined capacítatíve pathways; the Na+/Ca 2+ exchanger can also produce in sorne círcumstances net ínflux of Ca2+ (Míller, 1991; Clapham, 1995). Ca2+ ef­ flux to the extracellular space occurs against electrochemical gradíents, and thereby the pumpíng out of Ca2+ is directly (Caz+ pump) or indirectly (Na+/Ca2+) coupled to the hy­ drolysis of ATP.

1. Cytosolic free calcium ion concentration ([Ca2+]i) and whole-cell L-type Ca2+ channel currents were measured during excitation-contraction (E-C) coupling in single voltage-clamped rat cardiac ventricular cells. The measurements were used to compute the total cellular efflux of calcium ions through sarcoplasmic reticulum (SR) Ca2+ release channels (FSR,rel) and the influx of Ca2+ via L-type Ca2+ channels (FICa). 2. FSR,rel was elicited by depolarizing voltage-clamp pulses 200 ms in duration to membrane potentials from -30 to +80 mV. Over this range, peak FSR,rel had a bell-shaped dependence on clamp pulse potential. In all cells, the 'gain' of the system, measured as the ratio, FSR,rel(max)/FICa(max), declined from about 16, at 0 mV, to much lower values as clamp pulse voltage was made progressively more positive. We named this phenomenon of change in gain as a function of membrane potential, 'variable gain'. At clamp pulse potentials in the range -30 to 0 mV, the gain differed from cell to cell, being constant at about 16 in some cells, but decreasing from high values (approximately 65) at -20 mV in others. 3. At clamp pulse potentials at which Ca2+ influx (FICa) was maintained, FSR,rel also had a small maintained component. When macroscopic Ca2+ influx was brief (1-2 ms, during 'tails' of FICa), FSR,rel rose rapidly to a peak after repolarization and then declined with a half-time of about 9 ms (typically). 4. The rising phase of [Ca2+]i transients could be interrupted by stopping Ca2+ influx rapidly (by voltage clamp). We therefore termed this phenomenon 'interrupted SR Ca2+ release'.(ABSTRACT TRUNCATED AT 250 WORDS)

Producción Científica, The carotid body (CB) was defined as a sensory organ by De Castro in 1928. Two years later, Heymanns and coworkers demostrated that the organ was sensitive to alterations in blood gases and pH, in such a way that a decrease in blood P02 or pH or an increase in blood PC02 produced activation of the CB and, reflexely, hyperventilation. De Castro postulated that glomus cells were the sensor structures and that they should release sorn substance to transmit the stimulus to the sensory nerve endings (De Castro, 1928). De Castro's point of view, was widely accepted, and therefore the CB was considered a secondary sensory receptor. As a consequence, the principal aims of many workers in the chemoreception field have been to define the nature of the sensing mechanims ( sensory transduction process ) and to identify the substances released by chern cells.

Producción Científica, Carotid body (CB) chemoreceptors respond to a decrease in arterial blood pOz with an increase in action potential frequency in the sensory fibers innervating the organ, the response developing in a few seconds [l]. Chemoreceptor cells of the CB are the Oz sensing structures, releasing neurotransmitters in proportion to the pOz decrease and producing an electrical discharge of parallel intensity in the sensory nerve fibers of the carotid sinus nerve [2,3]. Recently, it has been shown that chemoreceptor cells possess an O,-sensitive K’ current that is reversibly inhibited by decreasing pOz in the bathing solution. The suggestion was made that this inhibition can lead to cell depolarization and activation of voltage-dependent Ca” channels [4,5]; the entry of Ca”+ through these channels, that are dihydropyridinesensitive, would trigger the release of neurotransmitters [6,7]. To accept this sequence, the inhibition of the K’ current must precede the activation of the sensory nerve discharges [8], that is, the inhibition mus: develop within the initial 3 s after lowering pOz, the time elapsing between a decrease in arterial pO1! and the increase in the carotid sinus nerve action potential frequency [l]. On the other hand, the suggestion has been made that the O?-sensor in chemoreceptor cells should be a hemoglobin- like hemoprotein, but no direct evidence to support this suggestion is available [l]. The presentstudy compares the time course of the inhibition of the K’ current by low pO1 and that of Na” current inhibition by TTX, which is known to occur in a few hundred ms [SJ. I: is shown that the former inhibition is faster. It is also shown that carbon monoxide, a very inert gas that in biological systems only reacts with hemoproteins, prevents the low PO,-induced inhibition of chemorcceptor cells E;” current.

1. Cat carotid bodies were incubated with the precursor [3H]tyrosine to label the catecholamine deposits and then mounted in a superfusion chamber which allowed simultaneous collection of the released [3H]dopamine (DA) and recording of action potentials from the carotid sinus nerve. 2. Low pH (7.2-6.6) superfusion of the carotid bodies for periods of 10 min produced a parallel increase in the release of [3H]DA and chemoreceptor discharge. 3. Carotid sinus nerve denervation of the carotid body 12-15 days prior to the experiments did not modify the release of [3H]DA elicited by low pH. 4. Superfusion of the carotid bodies with Ca(2+)-free, high-Mg2+ (1.6 mM) media reduced basal release of [3H]DA and chemoreceptor discharge by about 30%. Release evoked by low pH was reduced by 82%. Peak and average chemoreceptor discharge recorded in response to low pH were reduced by 28%. 5. Solutions containing weak acids (sodium acetate, 10 mM), adjusted at pH 7.4, elicited release of [3H]DA and increased chemoreceptor discharge. 6. With HCO3-CO2-buffered superfusion media, a reduction of bicarbonate to 5.6 mM (pH 6.8), an increase in CO2 to 20% (pH 6.8), or a simultaneous increase in CO2 to 20% and bicarbonate to 90 mM (pH 7.4), resulted in all cases in a corresponding increase in [3H]DA release and chemoreceptor discharge. The most effective stimulus was 20% CO2-pH 6.8 and the least effective 5% CO2-5.6 mM-HCO3-pH 6.8. 7. Inhibition of carbonic anhydrase with acetazolamide while perfusing the carotid bodies with a 20% CO2-equilibrated (pH 7.4) solution resulted in comparable reductions in the release of [3H]DA and chemoreceptor discharge. 8. It is concluded that the effective acidic stimulus at the carotid body chemoreceptors is an increase in hydrogen ion concentration in type I cells. It is also concluded that DA plays a critical role in the genesis of carotid sinus nerve discharges.

The ionic currents of carotid body type I cells and their possible involvement in the detection of oxygen tension (Po2) in arterial blood are unknown. The electrical properties of these cells were studied with the whole-cell patch clamp technique, and the hypothesis that ionic conductances can be altered by changes in PO2 was tested. The results show that type I cells have voltage-dependent sodium, calcium, and potassium channels. Sodium and calcium currents were unaffected by a decrease in PO2 from 150 to 10 millimeters of mercury, whereas, with the same experimental protocol, potassium currents were reversibly reduced by 25 to 50 percent. The effect of hypoxia was independent of internal adenosine triphosphate and calcium. Thus, ionic conductances, and particularly the O2-sensitive potassium current, play a key role in the transduction mechanism of arterial chemoreceptors.

Producción Científica, Hypoxia initiates the neurosecretory response of the carotid body (CB) by inhibiting one or more potassium channels in the chemoreceptor cells. Oxygen-sensitive K+ channels were first described in rabbit CB chemoreceptor cells, in which a transient outward K+ current was reported to be reversibly inhibited by hypoxia. Although progress has been made to characterize this current with electrophysiological and pharmacological tools, no attempts have been made to identify which Kv channel proteins are expressed in rabbit CB chemoreceptor cells and to determine + with adenoviruses that enabled ecdysone-inducible expression of the dominant-negative constructs and reporter genes in poly- cistronic vectors. In voltage-clamp experiments, we found that, whereas adenoviral infections of chemoreceptor cells with Kv1.xDN did not modify the O -sensitive K+ current, infections with Kv4.xDN suppressed the transient outward current in a time-dependent manner, significantly depolarized the cells, and abolished the depolarization induced by hypoxia. Our work dem- onstrate that genes of the Shal K+ channels underlie the tran- + their contribution to the native O2-sensitive K current. To probe sient outward, O2-sensitive, K current of rabbit CB chemore- the molecular identity of this current, we have used dominant- negative constructs to block the expression of functional Kv channels of the Shaker (Kv1.xDN) or the Shal (Kv4.xDN) subfam- ceptor cells and that this current contributes to the cell depolarization in response to low pO2. + ilies, because members of these two subfamilies contribute to Key words: O2-sensitive K current; viral gene transfer; the transient outward K+ currents in other preparations. Delivery of the constructs into chemoreceptor cells has been achieved, 2018-03-27

Short-term cell cultures were obtained from enzymatically dissociated carotid bodies from adult rabbits, and morphological and functional characterization of the cultured chemoreceptor cells were carried out. Under phase contrast, freshly isolated type I cells are round, bright, and 10-14 microns in diameter and exhibit strong fluorescence when stained with the glyoxylic acid technique. The content of endogenous dopamine in the cultures increased from 80 pmol/10(5) cells 2 h after plating the cells to 200 pmol/10(5) cells on the 3rd day, and the rate of synthesis and storage of [3H]dopamine from the precursor [3H]tyrosine increased from 1.7 pmol.10(5) cells-1.h-1 in 1-day cultures to 4 pmol.10(5) cells-1.h-1 on the 3rd day; the later values represent 80-85% of the expected values for the intact carotid body. After labeling with [3H]tyrosine, cultured chemoreceptor cells release [3H]dopamine when challenged by hypoxia, high external K+, or the protonophore dinitrophenol, the pattern of response being similar to that of the intact carotid body. When studied by whole cell clamp recording, individual chemoreceptor cells exhibit a marked variability in the properties of some ionic currents; the data, however, do not support the existence of distinct subpopulations of type I cells.

The plasticity of vascular smooth muscle cells that can switch from a contractile to a proliferative phenotype associates with several cardiovascular diseases. We have previously demonstrated that changes in the expression of voltage-dependent potassium (Kv) channels associated with this proliferative phenotype. These results were validated in transfected HEK293 cells: Kv1.5 decreased proliferation and Kv1.3 increased cell proliferation independently of ion flux (Cidad et al. ATVB 2012, 32:1299-307). Our data also suggested that the movement of the voltage-sensor of Kv1.3 channels could be coupled to signaling pathways leading to proliferation.Here we explore Kv1.3 regions involved in the signaling mechanism(s) associated with proliferation, by constructing chimeric Kv1.3-Kv1.5 channels and point-mutant Kv1.3 channels without consensus phosphorylation sites. These constructs were expressed as GFP- or cherry-fusion proteins, and we studied their functional expression, their effects on proliferation and their phosphorylation state in HEK cells combining immunocytochemical, electrophysiological and EdU incorporation studies.The molecular determinants of Kv1.3-induced proliferation are located at the carboxy-terminal domain. We identified two close residues (Y447 and S459) whose mutation abolished proliferation. Moreover, proliferation was increased with chimeric Kv1.5 mutants containing this segment (YS segment) but only when located at specific place within the channel molecule. Besides the selective MEK/ERK blocker PD98059 was able to inhibit Kv1.3-induced proliferation and tyrosine phosphorylation of the channel, having no effect on Y447A mutant channel. Finally, voltage-dependent transitions of Kv1.3 from close to open conformation increased pTyr labelling.We conclude that the signaling pathway linking Kv1.3 channels with proliferation involves voltage-induced conformational changes promoting tyrosine phosphorylation. Both phosphorylation and proliferation increase by facilitating the closed to open transition, being diminished in mutant channels that reside in the inactivated state. Supported by ISCIII-FEDER RD12/0042/0006, and BFU2013-45867-R grants.