Structural Determinants of Kv7.5 Potassium Channels That Confer Changes in Phosphatidylinositol 4,5-Bisphosphate (PIP2) Affinity and Signaling Sensitivities in Smooth Muscle Cells.

Smooth muscle cells express Kv7.4 and Kv7.5 voltage-dependent potassium channels, which have each been implicated as regulators of smooth muscle contractility, though they display different sensitivities to signaling via cAMP/protein kinase A (PKA) and protein kinase C (PKC). We expressed chimeric channels composed of different components of the Kv7.4 and Kv7.5 α-subunits in vascular smooth muscle cells to determine which components are essential for enhancement or inhibition of channel activity. Forskolin, an activator of the cAMP/PKA pathway, increased wild-type Kv7.5 but not wild-type Kv7.4 current amplitude. Replacing the amino terminus of Kv7.4 with the amino terminus of Kv7.5 conferred partial responsiveness to forskolin. In contrast, swapping carboxy-terminal phosphatidylinositol 4,5-bisphosphate (PIP2) binding domains, or the entire C terminus, was without effect on the forskolin response, but the latter conferred responsiveness to arginine-vasopressin (an inhibitory PKC-dependent response). Serine-to-alanine mutation at position 53 of the Kv7.5 amino terminus abrogated its ability to confer forskolin sensitivity to Kv7.4. Forskolin treatment reduced the sensitivity of Kv7.5 channels to Ciona intestinalis voltage-sensing phosphatase (Ci-VSP)-induced PIP2 depletion, whereas activation of PKC with phorbol-12-myristate-13-acetate potentiated the Ci-VSP-induced decline in Kv7.5 current amplitude. Our findings suggest that PKA-dependent phosphorylation of serine 53 on the amino terminus of Kv7.5 increases its affinity for PIP2, whereas PKC-dependent phosphorylation of the Kv7.5 carboxy terminus is associated with a reduction in PIP2 affinity; these changes in PIP2 affinity have corresponding effects on channel activity. Resting affinities for PIP2 differ for Kv7.4 and Kv7.5 based on differential responsiveness to Ci-VSP activation and different rates of current rundown in ruptured patch recordings. SIGNIFICANCE STATEMENT: Kv7.4 and Kv7.5 channels are known signal transduction intermediates and drug targets for regulation of smooth muscle tone. The present studies identify distinct functional domains that confer differential sensitivities of Kv7.4 and Kv7.5 to stimulatory and inhibitory signaling and reveal structural features of the channel subunits that determine their biophysical properties. These findings may improve our understanding of the roles of these channels in smooth muscle physiology and disease, particularly in conditions where Kv7.4 and Kv7.5 are differentially expressed.

Mechanisms of PKA-Dependent Potentiation of Kv7.5 Channel Activity in Human Airway Smooth Muscle Cells.

ß-adrenergic receptor (ßAR) activation promotes relaxation of both vascular and airway smooth muscle cells (VSMCs and ASMCs, respectively), though the signaling mechanisms have not been fully elucidated. We previously found that the activity of Kv7.5 voltage-activated potassium channels in VSMCs is robustly enhanced by activation of ßARs via a mechanism involving protein kinase A (PKA)-dependent phosphorylation. We also found that enhancement of Kv7 channel activity in ASMCs promotes airway relaxation. Here we provide evidence that Kv7.5 channels are natively expressed in primary cultures of human ASMCs and that they conduct currents which are robustly enhanced in response to activation of the ßAR/cyclic adenosine monophosphate (cAMP)/PKA pathway. MIT Scansite software analysis of putative PKA phosphorylation sites on Kv7.5 identified 8 candidate serine or threonine residues. Each residue was individually mutated to an alanine to prevent its phosphorylation and then tested for responses to ßAR activation or to stimuli that elevate cAMP levels. Only the mutation of serine 53 (S53A), located on the amino terminus of Kv7.5, significantly reduced the increase in Kv7.5 current in response to these stimuli. A phospho-mimic mutation (S53D) exhibited characteristics of ßAR-activated Kv7.5. Serine-to-alanine mutations of 6 putative PKA phosphorylation sites on the Kv7.5 C-terminus, individually or in combination, did not significantly reduce the enhancement of the currents in response to forskolin treatment (to elevate cAMP levels). We conclude that phosphorylation of S53 on the amino terminus of Kv7.5 is essential for PKA-dependent enhancement of channel activity in response to ßAR activation in vascular and airway smooth muscle cells.

PKC-dependent regulation of Kv7.5 channels by the bronchoconstrictor histamine in human airway smooth muscle cells.

Kv7 potassium channels have recently been found to be expressed and functionally important for relaxation of airway smooth muscle. Previous research suggests that native Kv7 currents are inhibited following treatment of freshly isolated airway smooth muscle cells with bronchoconstrictor agonists, and in intact airways inhibition of Kv7 channels is sufficient to induce bronchiolar constriction. However, the mechanism by which Kv7 currents are inhibited by bronchoconstrictor agonists has yet to be elucidated. In the present study, native Kv7 currents in cultured human trachealis smooth muscle cells (HTSMCs) were observed to be inhibited upon treatment with histamine; inhibition of Kv7 currents was associated with membrane depolarization and an increase in cytosolic Ca2+ ([Ca2+]cyt). The latter response was inhibited by verapamil, a blocker of L-type voltage-sensitive Ca2+ channels (VSCCs). Protein kinase C (PKC) has been implicated as a mediator of bronchoconstrictor actions, although the targets of PKC are not clearly established. We found that histamine treatment significantly and dose-dependently suppressed currents through overexpressed wild-type human Kv7.5 (hKv7.5) channels in cultured HTSMCs, and this effect was inhibited by the PKC inhibitor Ro-31-8220 (3 µM). The PKC-dependent suppression of hKv7.5 currents corresponded with a PKC-dependent increase in hKv7.5 channel phosphorylation. Knocking down or inhibiting PKCα, or mutating hKv7.5 serine 441 to alanine, abolished the inhibitory effects of histamine on hKv7.5 currents. These findings provide the first evidence linking PKC activation to suppression of Kv7 currents, membrane depolarization, and Ca2+ influx via L-type VSCCs as a mechanism for histamine-induced bronchoconstriction.

Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents.

Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth muscle cell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K(+) currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth muscle cells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled ß-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKA-dependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4.

Differential protein kinase C-dependent modulation of Kv7.4 and Kv7.5 subunits of vascular Kv7 channels.

The Kv7 family (Kv7.1-7.5) of voltage-activated potassium channels contributes to the maintenance of resting membrane potential in excitable cells. Previously, we provided pharmacological and electrophysiological evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels and that Kv7 activity is regulated by protein kinase C (PKC) in response to vasoconstrictors in vascular smooth muscle cells. Direct evidence for Kv7.4/7.5 heteromer formation, however, is lacking. Furthermore, it remains to be determined whether both subunits are regulated by PKC. Utilizing proximity ligation assays to visualize single molecule interactions, we now show that Kv7.4/Kv.7.5 heteromers are endogenously expressed in vascular smooth muscle cells. Introduction of dominant-negative Kv7.4 and Kv7.5 subunits in mesenteric artery myocytes reduced endogenous Kv7 currents by 84 and 76%, respectively. Expression of an inducible protein kinase Cα (PKCα) translocation system revealed that PKCα activation is sufficient to suppress endogenous Kv7 currents in A7r5 rat aortic and mesenteric artery smooth muscle cells. Arginine vasopressin (100 and 500 pm) and the PKC activator phorbol 12-myristate 13-acetate (1 nm) each inhibited human (h) Kv7.5 and hKv7.4/7.5, but not hKv7.4 channels expressed in A7r5 cells. A decrease in hKv7.5 and hKv7.4/7.5 current densities was associated with an increase in PKC-dependent phosphorylation of the channel proteins. These findings provide further evidence for a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation and new mechanistic insights into the role of heteromeric subunit assembly for regulation of vascular Kv7 channels.

Differential activation of vascular smooth muscle Kv7.4, Kv7.5, and Kv7.4/7.5 channels by ML213 and ICA-069673.

Recent research suggests that smooth muscle cells express Kv7.4 and Kv7.5 voltage-activated potassium channels, which contribute to maintenance of their resting membrane voltage. New pharmacologic activators of Kv7 channels, ML213 (N-mesitybicyclo[2.2.1]heptane-2-carboxamide) and ICA-069673 N-(6-chloropyridin-3-yl)-3,4-difluorobenzamide), have been reported to discriminate among channels formed from different Kv7 subtypes. We compared the effects of ML213 and ICA-069673 on homomeric human Kv7.4, Kv7.5, and heteromeric Kv7.4/7.5 channels exogenously expressed in A7r5 vascular smooth muscle cells. We found that, despite its previous description as a selective activator of Kv7.2 and Kv7.4, ML213 significantly increased the maximum conductance of homomeric Kv7.4 and Kv7.5, as well as heteromeric Kv7.4/7.5 channels, and induced a negative shift of their activation curves. Current deactivation rates decreased in the presence of the ML213 (10 µM) for all three channel combinations. Mutants of Kv7.4 (W242L) and Kv7.5 (W235L), previously found to be insensitive to another Kv7 channel activator, retigabine, were also insensitive to ML213 (10 µM). In contrast to ML213, ICA-069673 robustly activated Kv7.4 channels but was significantly less effective on homomeric Kv7.5 channels. Heteromeric Kv7.4/7.5 channels displayed intermediate responses to ICA-069673. In each case, ICA-069673 induced a negative shift of the activation curves without significantly increasing maximal conductance. Current deactivation rates decreased in the presence of ICA-069673 in a subunit-specific manner. Kv7.4 W242L responded to ICA-069673-like wild-type Kv7.4, but a Kv7.4 F143A mutant was much less sensitive to ICA-069673. Based on these results, ML213 and ICA-069673 likely bind to different sites and are differentially selective among Kv7.4, Kv7.5, and Kv7.4/7.5 channel subtypes.

KCNQ (Kv7) potassium channel activators as bronchodilators: combination with a ß2-adrenergic agonist enhances relaxation of rat airways.

KCNQ (Kv7 family) potassium (K(+)) channels were recently found in airway smooth muscle cells (ASMCs) from rodent and human bronchioles. In the present study, we evaluated expression of KCNQ channels and their role in constriction/relaxation of rat airways. Real-time RT-PCR analysis revealed expression of KCNQ4 > KCNQ5 > KCNQ1 > KCNQ2 > KCNQ3, and patch-clamp electrophysiology detected KCNQ currents in rat ASMCs. In precision-cut lung slices, the KCNQ channel activator retigabine induced a concentration-dependent relaxation of small bronchioles preconstricted with methacholine (MeCh; EC50 = 3.6 ± 0.3 µM). Bronchoconstriction was also attenuated in the presence of two other structurally unrelated KCNQ channel activators: zinc pyrithione (ZnPyr; 1 µM; 22 ± 7%) and 2,5-dimethylcelecoxib (10 µM; 24 ± 8%). The same three KCNQ channel activators increased KCNQ currents in ASMCs by two- to threefold. The bronchorelaxant effects of retigabine and ZnPyr were prevented by inclusion of the KCNQ channel blocker XE991. A long-acting ß2-adrenergic receptor agonist, formoterol (10 nM), did not increase KCNQ current amplitude in ASMCs, but formoterol (1-1,000 nM) did induce a time- and concentration-dependent relaxation of rat airways, with a notable desensitization during a 30-min treatment or with repetitive treatments. Coadministration of retigabine (10 µM) with formoterol produced a greater peak and sustained reduction of MeCh-induced bronchoconstriction and reduced the apparent desensitization observed with formoterol alone. Our findings support a role for KCNQ K(+) channels in the regulation of airway diameter. A combination of a ß2-adrenergic receptor agonist with a KCNQ channel activator may improve bronchodilator therapy.

Caveolin-3 regulates protein kinase A modulation of the Ca(V)3.2 (alpha1H) T-type Ca2+ channels.

Voltage-gated T-type Ca(2+) channel Ca(v)3.2 (α(1H)) subunit, responsible for T-type Ca(2+) current, is expressed in different tissues and participates in Ca(2+) entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Ca(v)3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca(2+) in different cells. We examined the localization and regulation of the Ca(v)3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Ca(v)3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Ca(v)3.1 and Ca(v)3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Ca(v)3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Ca(v)3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Ca(v)3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (I(Ca,T)) and adenovirus (AdCa(v)3.2)-mediated increase in peak Ca(v)3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of I(Ca,T) by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Ca(v)3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca(2+) signaling during normal and pathological processes.

Kv7 potassium channels in airway smooth muscle cells: signal transduction intermediates and pharmacological targets for bronchodilator therapy.

Expression and function of Kv7 (KCNQ) voltage-activated potassium channels in guinea pig and human airway smooth muscle cells (ASMCs) were investigated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), patch-clamp electrophysiology, and precision-cut lung slices. qRT-PCR revealed expression of multiple KCNQ genes in both guinea pig and human ASMCs. Currents with electrophysiological and pharmacological characteristics of Kv7 currents were measured in freshly isolated guinea pig and human ASMCs. In guinea pig ASMCs, Kv7 currents were significantly suppressed by application of the bronchoconstrictor agonists methacholine (100 nM) or histamine (30 µM), but current amplitudes were restored by addition of a Kv7 channel activator, flupirtine (10 µM). Kv7 currents in guinea pig ASMCs were also significantly enhanced by another Kv7.2-7.5 channel activator, retigabine, and by celecoxib and 2,5-dimethyl celecoxib. In precision-cut human lung slices, constriction of airways by histamine was significantly reduced in the presence of flupirtine. Kv7 currents in both guinea pig and human ASMCs were inhibited by the Kv7 channel blocker XE991. In human lung slices, XE991 induced robust airway constriction, which was completely reversed by addition of the calcium channel blocker verapamil. These findings suggest that Kv7 channels in ASMCs play an essential role in the regulation of airway diameter and may be targeted pharmacologically to relieve airway hyperconstriction induced by elevated concentrations of bronchoconstrictor agonists.

Regulation and function of Cav3.1 T-type calcium channels in IGF-I-stimulated pulmonary artery smooth muscle cells.

Arterial smooth muscle cells enter the cell cycle and proliferate in conditions of disease and injury, leading to adverse vessel remodeling. In the pulmonary vasculature, diverse stimuli cause proliferation of pulmonary artery smooth muscle cells (PASMCs), pulmonary artery remodeling, and the clinical condition of pulmonary hypertension associated with significant health consequences. PASMC proliferation requires extracellular Ca(2+) influx that is intimately linked with intracellular Ca(2+) homeostasis. Among the primary sources of Ca(2+) influx in PASMCs is the low-voltage-activated family of T-type Ca(2+) channels; however, up to now, mechanisms for the action of T-type channels in vascular smooth muscle cell proliferation have not been addressed. The Ca(v)3.1 T-type Ca(2+) channel mRNA is upregulated in cultured PASMCs stimulated to proliferate with insulin-like growth factor-I (IGF-I), and this upregulation depends on phosphatidylinositol 3-kinase/Akt signaling. Multiple stimuli that trigger an acute rise in intracellular Ca(2+) in PASMCs, including IGF-I, also require the expression of Ca(v)3.1 Ca(2+) channels for their action. IGF-I also led to cell cycle initiation and proliferation of PASMCs, and, when expression of the Ca(v)3.1 Ca(2+) channel was knocked down by RNA interference, so were the expression and activation of cyclin D, which are necessary steps for cell cycle progression. These results confirm the importance of T-type Ca(2+) channels in proper progression of the cell cycle in PASMCs stimulated to proliferate by IGF-I and suggest that Ca(2+) entry through Ca(v)3.1 T-type channels in particular interacts with Ca(2+)-dependent steps of the mitogenic signaling cascade as a central component of vascular remodeling in disease.

Diclofenac distinguishes among homomeric and heteromeric potassium channels composed of KCNQ4 and KCNQ5 subunits.

KCNQ4 and KCNQ5 potassium channel subunits are expressed in vascular smooth muscle cells, although it remains uncertain how these subunits assemble to form functional channels. Using patch-clamp techniques, we compared the electrophysiological characteristics and effects of diclofenac, a known KCNQ channel activator, on human KCNQ4 and KCNQ5 channels expressed individually or together in A7r5 rat aortic smooth muscle cells. The conductance curves of the overexpressed channels were fitted by a single Boltzmann function in each case (V(0.5) values: -31, -44, and -38 mV for KCNQ4, KCNQ5, and KCNQ4/5, respectively). Diclofenac (100 µM) inhibited KCNQ5 channels, reducing maximum conductance by 53%, but increased maximum conductance of KCNQ4 channels by 38%. The opposite effects of diclofenac on KCNQ4 and KCNQ5 could not be attributed to the presence of a basic residue (lysine) in the voltage-sensing domain of KCNQ5, because mutation of this residue to neutral glycine (the residue present in KCNQ4) resulted in a more effective block of the channel. Differences in deactivation rates and distinct voltage-dependent effects of diclofenac on channel activation and deactivation observed with each of the subunit combinations (KCNQ4, KCNQ5, and KCNQ4/5) were used as diagnostic tools to evaluate native KCNQ currents in vascular smooth muscle cells. A7r5 cells express only KCNQ5 channels endogenously, and their responses to diclofenac closely resembled those of the overexpressed KCNQ5 currents. In contrast, mesenteric artery myocytes, which express both KCNQ4 and KCNQ5 channels, displayed whole-cell KCNQ currents with properties and diclofenac responses characteristic of overexpressed heteromeric KCNQ4/5 channels.

T-type voltage-gated calcium channels regulate the tone of mouse efferent arterioles.

Voltage-gated calcium channels are important for the regulation of renal blood flow and the glomerular filtration rate. Excitation-contraction coupling in afferent arterioles is known to require activation of these channels and we studied their role in the regulation of cortical efferent arteriolar tone. We used microdissected perfused mouse efferent arterioles and found a transient vasoconstriction in response to depolarization with potassium; an effect abolished by removal of extracellular calcium. The T-type voltage-gated calcium channel antagonists mibefradil and nickel blocked this potassium-induced constriction. Further, constriction by the thromboxane analogue U46619 was significantly inhibited by mibefradil at a concentration specific for T-type channels. Using PCR, we found that two channel subtypes, Ca(v)3.1 and Ca(v)3.2, were expressed in microdissected efferent arterioles. Ca(v)3.1 was found by immunocytochemistry to be located in mouse efferent arterioles, human pre- and postglomerular vasculature, and Ca(v)3.2 in rat glomerular arterioles. Inhibition of endothelial nitric oxide synthase by L-NAME or its deletion by gene knockout changed the potassium-elicited transient constriction to a sustained response. Low concentrations of nickel, an agent that blocks Ca(v)3.2, had a similar effect. Thus, T-type voltage-gated calcium channels are functionally important for depolarization-induced vasoconstriction and subsequent dilatation in mouse cortical efferent arterioles.

Heteromeric Channels Formed From Alternating Kv7.4 and Kv7.5 α-Subunits Display Biophysical, Regulatory, and Pharmacological Characteristics of Smooth Muscle M-Currents.

Smooth muscle cells of the vasculature, viscera, and lungs generally express multiple α-subunits of the Kv7 voltage-gated potassium channel family, with increasing evidence that both Kv7.4 and Kv7.5 can conduct "M-currents" that are functionally important for the regulation of smooth muscle contractility. Although expression systems demonstrate that functional channels can form as homomeric tetramers of either Kv7.4 or Kv7.5 α-subunits, there is evidence that heteromeric channel complexes, containing some combination of Kv7.4 and Kv7.5 α-subunits, may represent the predominant configuration natively expressed in some arterial myocytes, such as rat mesenteric artery smooth muscle cells (MASMCs). Our previous work has suggested that Kv7.4/Kv7.5 heteromers can be distinguished from Kv7.4 or Kv7.5 homomers based on their biophysical, regulatory, and pharmacological characteristics, but it remains to be determined how Kv7.4 and Kv7.5 α-subunits combine to produce these distinct characteristics. In the present study, we constructed concatenated dimers or tetramers of Kv7.4 and Kv7.5 α-subunits and expressed them in a smooth muscle cell line to determine if a particular α-subunit configuration can exhibit the features previously reported for natively expressed Kv7 currents in MASMCs. Several unique characteristics of native smooth muscle M-currents were reproduced under conditions that constrain channel formation to a Kv7.4:Kv7.5 stoichiometry of 2:2, with alternating Kv7.4 and Kv7.5 α-subunits within a tetrameric structure. Although other subunit arrangements/combinations are not ruled out, the findings provide new insights into the oligomerization of α-subunits and the ways in which Kv7.4/Kv7.5 subunit assembly can affect smooth muscle signal transduction and pharmacological responses to Kv7 channel modulating drugs.

Differential effects of selective cyclooxygenase-2 inhibitors on vascular smooth muscle ion channels may account for differences in cardiovascular risk profiles.

Celecoxib, rofecoxib, and diclofenac are clinically used cyclooxygenase-2 (COX-2) inhibitors, which have been under intense scrutiny because long-term rofecoxib (Vioxx; Merck, Whitehouse Station, NJ) treatment was found to increase the risk of adverse cardiovascular events. A differential risk profile for these drugs has emerged, but the underlying mechanisms have not been fully elucidated. We investigated the effects of celecoxib, rofecoxib, and diclofenac on ionic currents and calcium signaling in vascular smooth muscle cells (VSMCs) using patch-clamp techniques and fura-2 fluorescence and on arterial constriction using pressure myography. Celecoxib, but not rofecoxib or diclofenac, dramatically enhanced KCNQ (K(v)7) potassium currents and suppressed L-type voltage-sensitive calcium currents in A7r5 rat aortic smooth muscle cells (native KCNQ currents or overexpressed human KCNQ5 currents) and freshly isolated rat mesenteric artery myocytes. The effects of celecoxib were concentration-dependent within the therapeutic concentration range, and were reversed on washout. Celecoxib, but not rofecoxib, also inhibited calcium responses to vasopressin in A7r5 cells and dilated intact or endothelium-denuded rat mesenteric arteries. A celecoxib analog, 2,5-dimethyl-celecoxib, which does not inhibit COX-2, mimicked celecoxib in its enhancement of vascular KCNQ5 currents, suppression of L-type calcium currents, and vasodilation. We conclude that celecoxib inhibits calcium responses in VSMCs by enhancing KCNQ5 currents and suppressing L-type calcium currents, which ultimately reduces vascular tone. These effects are independent of its COX-2 inhibitory actions and may explain the differential risk of cardiovascular events in patients taking different drugs of this class.

T-type calcium channels are regulated by hypoxia/reoxygenation in ventricular myocytes.

Low-voltage-activated calcium channels are reexpressed in ventricular myocytes in pathological conditions associated with hypoxic episodes, but a direct relation between oxidative stress and T-type channel function and regulation in cardiomyocytes has not been established. We aimed to investigate low-voltage-activated channel regulation under oxidative stress in neonatal rat ventricular myocytes. RT-PCR measurements of voltage-gated Ca(2+) (Ca(v))3.1 and Ca(v)3.2 mRNA levels in oxidative stress were compared with whole cell patch-clamp recordings of T-type calcium current. The results indicate that hypoxia reduces T-type current density at -30 mV (the hallmark of this channel) based on the shift of the voltage dependence of activation to more depolarized values and downregulation of Ca(v)3.1 at the mRNA level. Upon reoxygenation, both Ca(v)3.1 mRNA levels and the voltage dependence of total T-type current are restored, although differently for activation and inactivation. Using Ni(2+), we distinguished different effects of hypoxia/reoxygenation on the two current components. Long-term incubation in the presence of 100 microM CoCl(2) reproduced the effects of hypoxia on T-type current activation and inactivation, indicating that the chemically induced oxidative state is sufficient to alter T-type calcium current activity, and that hypoxia-inducible factor-1alpha is involved in Ca(v)3.1 downregulation. Our results demonstrate that Ca(v)3.1 and Ca(v)3.2 T-type calcium channels are differentially regulated by hypoxia/reoxygenation injury, and, therefore, they may serve different functions in the myocyte in response to hypoxic injury.

Non-L-type voltage-dependent calcium channels control vascular tone of the rat basilar artery.

1. Constriction of cerebral arteries is considered to depend on L-type voltage-dependent calcium channels (VDCCs); however, many previous studies have used antagonists with potential non-selective actions. Our aim was to determine the expression and function of VDCCs in the rat basilar artery. 2. The relative expression of VDCC subtypes was assessed using quantitative polymerase chain reaction and immunohistochemistry. Data were correlated with physiological studies of vascular function. Domains I-II of the T channel subtypes expressed in the rat basilar artery were cloned and sequenced. 3. Blockade of L-type channels with nifedipine had no effect on vascular tone. In contrast, in the presence of nifedipine, hyperpolarization of short arterial segments produced relaxation, whereas depolarization of quiescent segments evoked constriction. 4. The mRNA and protein for L- and T-type VDCCs were strongly expressed in the main basilar artery and side branches, with Ca(V)3.1 and Ca(V)1.2 the predominant subtypes. 5. T-Type VDCC blockers (i.e. 1 micromol/L mibefradil, 10 micromol/L pimozide and 100 micromol/L flunarizine) decreased intracellular calcium in smooth muscle cells, relaxed and hyperpolarized arteries, whereas nickel chloride (100 micromol/L) had no effect. In contrast with nifedipine, 10 micromol/L nimodipine produced hyperpolarization and relaxation. 6. When arteries were relaxed with 10 micromol/L U73122 (a phospholipase C inhibitor) in the presence of nifedipine, 40 mmol/L KCl evoked depolarization and constriction, which was significantly reduced by 1 micromol/L mibefradil. 7. Sequencing of domains I-II revealed splice variants of Ca(V)3.1, which may impact on channel activity. 8. We conclude that vascular tone of the rat basilar artery results from calcium influx through nifedipine-insensitive VDCCs with pharmacology consistent with Ca(V)3.1 T-type channels.

Vascular KCNQ potassium channels as novel targets for the control of mesenteric artery constriction by vasopressin, based on studies in single cells, pressurized arteries, and in vivo measurements of mesenteric vascular resistance.

Pressor effects of the vasoconstrictor hormone arginine vasopressin (AVP), observed when systemic AVP concentrations are less than 100 pM, are important for the physiological maintenance of blood pressure, and they are also the basis for therapeutic use of vasopressin to restore blood pressure in hypotensive patients. However, the mechanisms by which circulating AVP induces arterial constriction are unclear. We examined the novel hypothesis that KCNQ potassium channels mediate the physiological vasoconstrictor actions of AVP. Reverse transcriptase polymerase chain reaction revealed expression of KCNQ1, KCNQ4, and KCNQ5 in rat mesenteric artery smooth muscle cells (MASMCs). Whole-cell perforated patch recordings of voltage-sensitive K+ (Kv) currents in freshly isolated MASMCs revealed 1,3-dihydro-1-phenyl-3,3-bis(4-pyridinylmethyl)-2H-indol-2-one (linopirdine)- and 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991)-sensitive KCNQ currents that were electrophysiologically and pharmacologically distinct from other Kv currents. Suppression of KCNQ currents by AVP (100 pM) was associated with significant membrane depolarization, and it was abolished by the protein kinase C (PKC) inhibitor calphostin C (250 nM). The KCNQ channel blocker linopirdine (10 microM) inhibited KCNQ currents in MASMCs, and it induced constriction of isolated rat mesenteric arteries. The vasoconstrictor responses were not additive when combined with 30 pM AVP, and they were prevented by the L-type Ca2+ channel blocker verapamil. Ethyl-N-[2-amino-6-(4-fluorophenylmethylamino)pyridin-3-yl] carbamic acid (flupirtine) significantly enhanced KCNQ currents, and it reversed constrictor responses to 30 pM AVP. In vivo, i.v. administration of linopirdine induced a dose-dependent increase in mesenteric artery resistance and blood pressure, whereas flupirtine had the opposite effects. We conclude that physiological concentrations of AVP induce mesenteric artery constriction via PKC-dependent suppression of KCNQ currents and L-type Ca2+ channel activation in MASMCs.

T-type Ca2+ channels in vascular smooth muscle: multiple functions.

Vascular smooth muscle is a major constituent of the blood vessel wall, and its many functions depend on type and location of the vessel, developmental or pathological state, and environmental and chemical factors. Vascular smooth muscle cells (VSMCs) use calcium as a signal molecule for multiple functions. An important component of calcium signaling pathways is the entry of extracellular calcium via voltage-gated Ca2+ channels, which in vascular smooth muscle cells (VSMCs) are of two main types, the high voltage-activated (HVA) L-type and low voltage-activated (LVA) T-type channels. Whereas L-type channels function primarily to regulate Ca2+ entry for contraction, it is generally accepted that T-type Ca2+ channels do not contribute significantly to arterial vasoconstriction, with the possible exception of the renal microcirculation. T-type Ca2+ channels are also present in some veins that display spontaneous contractile activity, where they likely generate pacemaker activity. T-type Ca2+ channel expression has also been associated with normal and pathological proliferation of VSMCs, often stimulated by external cues in response to insult or injury. Expression of T-type channels has been linked to the G1 and S phases of the cell cycle, a period important for the signaling of gene expression necessary for cell growth, progression of the cell cycle and ultimately cell division. To better understand T-type Ca2+ channel functions in VSM, it will be necessary to develop new approaches that are specifically targeted to this class of Ca2+ channels and its individual members.

Variation of T-type calcium channel protein expression affects cell division of cultured tumor cells.

In this study we investigated the T-type calcium channel and its involvement in the cell division of U87MG cultured glioma cells and N1E-115 neuroblastoma cells. Using Western blot analysis, we found that expression of both alpha1G and alpha1H subunits of the T-type calcium channel decreased during conditions associated with a decrease in proliferation as evidenced by increased expression of cyclin D1, a marker for non-proliferating cells. Both serum starvation and application of mibefradil, a selective T-type calcium channel antagonist, resulted in a 50% decrease in the expression of alpha1G and alpha1H and a 700-900% increase in levels of cyclin D1 in U87MG and N1E-115 cells, respectively. Furthermore, overexpression of the alpha1H subunit resulted in a two-fold increase in cell proliferation compared to control cultures or cultures receiving an empty vector. In contrast, blocking expression of the alpha1G subunit using antisense oligonucleotides lead to a 70% decrease in proliferation of U87MG and N1E-115 cells compared to control cultures or cultures receiving a scrambled oligonucleotide. Our findings suggest that proliferation of U87MG glioma cells and N1E-115 is regulated by T-type calcium channel expression.

Elimination of the actin-binding domain in kelch-like 1 protein induces T-type calcium channel modulation only in the presence of action potential waveforms.

The Kelch-like 1 protein (KLHL1) is a neuronal actin-binding protein that modulates calcium channel function. It increases the current density of Ca(v)3.2 (α(1H)) calcium channels via direct interaction with α(1H) and actin-F, resulting in biophysical changes in Ca(v)3.2 currents and an increase in recycling endosomal activity with subsequent increased α(1H) channel number at the plasma membrane. Interestingly, removal of the actin-binding Kelch motif (ΔKelch) prevents the increase in Ca(v)3.2 current density seen with wild-type KLHL1 when tested with normal square pulse protocols but does not preclude the effect when tested using action potential waveforms (AP). Here, we dissected the kinetic properties of the AP waveform that confer the mutant Kelch the ability to interact with Ca(v)3.2 and induce an increase in calcium influx. We modified the action potential waveform by altering the slopes of repolarization and/or recovery from hyperpolarization or by changing the duration of the depolarization plateau or the hyperpolarization phase and tested the modulation of Ca(v)3.2 by the mutant ΔKelch. Our results show that the recovery phase from hyperpolarization phase determines the conformational changes that allow the α(1H) subunit to properly interact with mutant KLHL1 lacking its actin-binding Kelch domains, leading to increased Ca influx.