The angiotensin II type 1 receptor (AT1R) closely interacts with large conductance voltage- and Ca2+-activated K+ (BK) channels and inhibits their activity independent of G-protein activation.

Angiotensin II (ANG-II) and BK channels play important roles in the regulation of blood pressure. In arterial smooth muscle, ANG-II inhibits BK channels, but the underlying molecular mechanisms are unknown. Here, we first investigated whether ANG-II utilizes its type 1 receptor (AT1R) to modulate BK activity. Pharmacological, biochemical, and molecular evidence supports a role for AT1R. In renal arterial myocytes, the AT1R antagonist losartan (10 µM) abolished the ANG-II (1 µM)-induced reduction of whole cell BK currents, and BK channels and ANG-II receptors were found to co-localize at the cell periphery. We also found that BK inhibition via ANG-II-activated AT1R was independent of G-protein activation (assessed with 500 µM GDPßS). In BK-expressing HEK293T cells, ANG-II (1 µM) also induced a reduction of BK currents, which was contingent on AT1R expression. The molecular mechanisms of AT1R and BK channel coupling were investigated in co-transfected cells. Co-immunoprecipitation showed formation of a macromolecular complex, and live immunolabeling demonstrated that both proteins co-localized at the plasma membrane with high proximity indexes as in arterial myocytes. Consistent with a close association, we discovered that the sole AT1R expression could decrease BK channel voltage sensitivity. Truncated BK proteins revealed that the voltage-sensing conduction cassette is sufficient for BK-AT1R association. Finally, C-terminal yellow and cyan fluorescent fusion proteins, AT1R-YFP and BK-CFP, displayed robust co-localized Förster resonance energy transfer, demonstrating intermolecular interactions at their C termini. Overall, our results strongly suggest that AT1R regulates BK channels through a close protein-protein interaction involving multiple BK regions and independent of G-protein activation.

The ß1-subunit of the MaxiK channel associates with the thromboxane A2 receptor and reduces thromboxane A2 functional effects.

The large conductance voltage- and Ca(2+)-activated K(+) channel (MaxiK, BK(Ca), BK) is composed of four pore-forming α-subunits and can be associated with regulatory ß-subunits. One of the functional roles of MaxiK is to regulate vascular tone. We recently found that the MaxiK channel from coronary smooth muscle is trans-inhibited by activation of the vasoconstricting thromboxane A(2) prostanoid receptor (TP), a mechanism supported by MaxiK α-subunit (MaxiKα)-TP physical interaction. Here, we examined the role of the MaxiK ß1-subunit in TP-MaxiK association. We found that the ß1-subunit can by itself interact with TP and that this association can occur independently of MaxiKα. Subcellular localization analysis revealed that ß1 and TP are closely associated at the cell periphery. The molecular mechanism of ß1-TP interaction involves predominantly the ß1 extracellular loop. As reported previously, TP activation by the thromboxane A(2) analog U46619 caused inhibition of MaxiKα macroscopic conductance or fractional open probability (FP(o)) as a function of voltage. However, the positive shift of the FP(o) versus voltage curve by U46619 relative to the control was less prominent when ß1 was coexpressed with TP and MaxiKα proteins (20 ± 6 mV, n = 7) than in cells expressing TP and MaxiKα alone (51 ± 7 mV, n = 7). Finally, ß1 gene ablation reduced the EC(50) of the U46619 agonist in mediating aortic contraction from 18 ± 1 nm (n = 12) to 9 ± 1 nm (n = 12). The results indicate that the ß1-subunit can form a tripartite complex with TP and MaxiKα, has the ability to associate with each protein independently, and diminishes U46619-induced MaxiK channel trans-inhibition as well as vasoconstriction.

The first transmembrane domain (TM1) of ß2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels.

The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary ß-subunit that modulates the voltage- and Ca(2+)-dependent activation of the channel. Structural components present in ß-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the ß2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein-protein interactions demonstrated for the first time that TM1 of the ß2-subunit physically binds to the transmembrane S1 domain of the α-subunit.

Unconventional myristoylation of large-conductance Ca²âº-activated K⁺ channel (Slo1) via serine/threonine residues regulates channel surface expression.

Protein myristoylation is a means by which cells anchor proteins into membranes. The most common type of myristoylation occurs at an N-terminal glycine. However, myristoylation rarely occurs at an internal amino acid residue. Here we tested whether the α-subunit of the human large-conductance voltage- and Ca(2+)-activated K(+) channel (hSlo1) might undergo internal myristoylation. hSlo1 expressed in HEK293T cells incorporated [(3)H]myristic acid via a posttranslational mechanism, which is insensitive to cycloheximide, an inhibitor of protein biosynthesis. In-gel hydrolysis of [(3)H]myristoyl-hSlo1 with alkaline NH(2)OH (which cleaves hydroxyesters) but not neutral NH(2)OH (which cleaves thioesters) completely removed [(3)H]myristate from hSlo1, suggesting the involvement of a hydroxyester bond between hSlo1's hydroxyl-bearing serine, threonine, and/or tyrosine residues and myristic acid; this type of esterification was further confirmed by its resistance to alkaline Tris·HCl. Treatment of cells expressing hSlo1 with 100 µM myristic acid caused alteration of hSlo1 activation kinetics and a 40% decrease in hSlo1 current density from 20 to 12 nA*MΩ. Immunocytochemistry confirmed a decrease in hSlo1 plasmalemma localization by myristic acid. Replacement of the six serines or the seven threonines (but not of the single tyrosine) of hSlo1 intracellular loops 1 and 3 with alanines decreased hSlo1 direct myristoylation by 40-44%, whereas in combination decreased myristoylation by nearly 90% and abolished the myristic acid-induced change in current density. Our data demonstrate that an ion channel, hSlo1, is internally and posttranslationally myristoylated. Myristoylation occurs mainly at hSlo1 intracellular loop 1 or 3, and is an additional mechanism for channel surface expression regulation.

Thromboxane A2 receptor and MaxiK-channel intimate interaction supports channel trans-inhibition independent of G-protein activation.

Large conductance voltage- and calcium-activated potassium channels (MaxiK, BK(Ca)) are well known for sustaining cerebral and coronary arterial tone and for their linkage to vasodilator ß-adrenergic receptors. However, how MaxiK channels are linked to counterbalancing vasoconstrictor receptors is unknown. Here, we show that vasopressive thromboxane A2 receptors (TP) can intimately couple with and inhibit MaxiK channels. Activation of the receptor with its agonist trans-inhibits MaxiK independently of G-protein activation. This unconventional mechanism is supported by independent lines of evidence: (i) inhibition of MaxiK current by thromboxane A2 mimetic, U46619, occurs even when G-protein activity is suppressed; (ii) MaxiK and TP physically associate and display a high degree of proximity; and (iii) Förster resonance energy transfer occurs between fluorescently labeled MaxiK and TP, supporting a direct interaction. The molecular mechanism of MaxiK-TP intimate interaction involves the receptor's first intracellular loop and C terminus, and it entails the voltage-sensing conduction cassette of MaxiK channel. Further, physiological evidence of MaxiK-TP physical interaction is given in human coronaries and rat aorta, and by confirming TP role (with antagonist SQ29,548) in the U46619-induced MaxiK inhibition in human coronaries. We propose that vasoconstrictor TP receptor and MaxiK-channel direct interaction facilitates G-protein-independent TP to MaxiK trans-inhibition, which would promote vasoconstriction.

Identification of a functional interaction between Kv4.3 channels and c-Src tyrosine kinase.

Voltage-gated K(+) (Kv) channels are key determinants of cardiac and neuronal excitability. A substantial body of evidence has accumulated in support of a role for Src family tyrosine kinases in the regulation of Kv channels. In this study, we examined the possibility that c-Src tyrosine kinase participates in the modulation of the transient voltage-dependent K(+) channel Kv4.3. Supporting a mechanistic link between Kv4.3 and c-Src, confocal microscopy analysis of HEK293 cells stably transfected with Kv4.3 showed high degree of co-localization of the two proteins at the plasma membrane. Our results further demonstrate an association between Kv4.3 and c-Src by co-immunoprecipitation and GST pull-down assays, this interaction being mediated by the SH2 and SH3 domains of c-Src. Furthermore, we show that Kv4.3 is tyrosine phosphorylated under basal conditions. The functional relevance of the observed interaction between Kv4.3 and c-Src was established in patch-clamp experiments, where application of the Src inhibitor PP2 caused a decrease in Kv4.3 peak current amplitude, but not the inactive structural analogue PP3. Conversely, intracellular application of recombinant c-Src kinase or the protein tyrosine phosphatase inhibitor bpV(phen) increased Kv4.3 peak current amplitude. In conclusion, our findings provide evidence that c-Src-induced Kv4.3 channel activation involves their association in a macromolecular complex and suggest a role for c-Src-Kv4.3 pathway in regulating cardiac and neuronal excitability.

c-Src tyrosine kinase, a critical component for 5-HT2A receptor-mediated contraction in rat aorta.

Serotonin (5-hydroxytryptamine, 5-HT) receptors (5-HTRs) play critical roles in brain and cardiovascular functions. In the vasculature, 5-HT induces potent vasoconstrictions, which in aorta are mainly mediated by activation of the 5-HT(2A)R subtype. We previously proposed that one signalling mechanism of 5-HT-induced vasoconstriction could be c-Src, a member of the Src tyrosine kinase family. We now provide evidence for a central role of c-Src in 5-HT(2A)R-mediated contraction. Inhibition of Src kinase activity with 10 mum 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) prior to contraction resulted in approximately 90-99% inhibition of contractions induced by 5-HT or by alpha-methyl-5-HT (5-HT(2)R agonist). In contrast, PP2 pretreatment only partly inhibited contractions induced by angiotensin II and the thromboxane A(2) mimetic, U46619, and had no significant action on phenylephrine-induced contractions. 5-Hydroxytryptamine increased Src kinase activity and PP2-sensitive tyrosine-phosphorylated proteins. As expected for c-Src identity, PP2 pretreatment inhibited 5-HT-induced contraction with an IC(50) of approximately 1 mum. Ketanserin (10 nm), a 5-HT(2A) antagonist, but not antagonists of 5-HT(2B)R (100 nm SB204741) or 5-HT(2C)R (20 nm RS102221), prevented 5-HT-induced contractions, mimicking PP2 and implicating 5-HT(2A)R as the major receptor subtype coupled to c-Src. In HEK 293T cells, c-Src and 5-HT(2A)R were reciprocally co-immunoprecipitated and co-localized at the cell periphery. Finally, 5-HT-induced Src activity was unaffected by inhibition of Rho kinase, supporting a role of c-Src upstream of Rho kinase. Together, the results highlight c-Src activation as one of the early and pivotal mechanisms in 5-HT(2A)R contractile signalling in aorta.

Slo1 caveolin-binding motif, a mechanism of caveolin-1-Slo1 interaction regulating Slo1 surface expression.

The large conductance, voltage- and Ca2+-activated potassium (MaxiK, BK) channel and caveolin-1 play important roles in regulating vascular contractility. Here, we hypothesized that the MaxiK alpha-subunit (Slo1) and caveolin-1 may interact with each other. Slo1 and caveolin-1 physiological association in native vascular tissue is strongly supported by (i) detergent-free purification of caveolin-1-rich domains demonstrating a pool of aortic Slo1 co-migrating with caveolin-1 to light density sucrose fractions, (ii) reverse co-immunoprecipitation, and (iii) double immunolabeling of freshly isolated myocytes revealing caveolin-1 and Slo1 proximity at the plasmalemma. In HEK293T cells, Slo1-caveolin-1 association was unaffected by the smooth muscle MaxiK beta1-subunit. Sequence analysis revealed two potential caveolin-binding motifs along the Slo1 C terminus, one equivalent, 1007YNMLCFGIY1015, and another mirror image, 537YTEYLSSAF545, to the consensus sequence, varphiXXXXvarphiXXvarphi. Deletion of 1007YNMLCFGIY1015 caused approximately 80% loss of Slo1-caveolin-1 association while preserving channel normal folding and overall Slo1 and caveolin-1 intracellular distribution patterns. 537YTEYLSSAF545 deletion had an insignificant dissociative effect. Interestingly, caveolin-1 coexpression reduced Slo1 surface and functional expression near 70% without affecting channel voltage sensitivity, and deletion of 1007YNMLCFGIY1015 motif obliterated channel surface expression. The results suggest 1007YNMLCFGIY1015 possible participation in Slo1 plasmalemmal targeting and demonstrate its role as a main mechanism for caveolin-1 association with Slo1 potentially serving a dual role: (i) maintaining channels in intracellular compartments downsizing their surface expression and/or (ii) serving as anchor of plasma membrane resident channels to caveolin-1-rich membranes. Because the caveolin-1 scaffolding domain is juxtamembrane, it is tempting to suggest that Slo1-caveolin-1 interaction facilitates the tethering of the Slo1 C-terminal end to the membrane.

Fenofibrate inhibits intestinal Cl- secretion by blocking basolateral KCNQ1 K+ channels.

Fibrates are peroxisome proliferator-activated receptor-alpha (PPARalpha) ligands in widespread clinical use to lower plasma triglyceride levels. We investigated the effect of fenofibrate and clofibrate on ion transport in mouse intestine and in human T84 colonic adenocarcinoma cells through the use of short-circuit current (I(sc)) and ion flux analysis. In mice, oral administration of fenofibrate produced a persistent inhibition of cAMP-stimulated electrogenic Cl(-) secretion by isolated jejunum and colon without affecting electroneutral fluxes of (22)Na(+) or (86)Rb(+) (K(+)) across unstimulated colonic mucosa. When applied acutely to isolated mouse intestinal mucosa, 100 microM fenofibrate inhibited cAMP-stimulated I(sc) within 5 min. In T84 cells, fenofibrate rapidly inhibited approximately 80% the Cl(-) secretory responses to forskolin (cAMP) and to heat stable enterotoxin STa (cGMP) without affecting the response to carbachol (Ca(2+)). Both fenofibrate and clofibrate inhibited cAMP-stimulated I(sc) with an IC(50) approximately 1 muM, whereas other PPARalpha activators (gemfibrozil and Wy-14,643) were without effect. Membrane permeabilization experiments on T84 cells indicated that fenofibrate inhibits basolateral cAMP-stimulated K(+) channels (putatively KCNQ1/KCNE3) without affecting Ca(2+)-stimulated K(+) channel activity, whereas clofibrate inhibits both K(+) pathways. Fenofibrate had no effect on apical cAMP-stimulated Cl(-) channel activity. Patch-clamp analysis of HEK-293T cells confirmed that 100 microM fenofibrate rapidly inhibits K(+) currents associated with ectopic expression of human KCNQ1 with or without the KCNE3 beta-subunit. We conclude that fenofibrate inhibits intestinal cAMP-stimulated Cl(-) secretion through a nongenomic mechanism that involves a selective inhibition of basolateral KCNQ1/KCNE3 channel complexes. Our findings raise the prospect of fenofibrate as a safe and effective antidiarrheal agent.

Regulation of mouse Slo gene expression: multiple promoters, transcription start sites, and genomic action of estrogen.

The large conductance, voltage- and Ca(2+)-activated K(+) channel plays key roles in diverse body functions influenced by estrogen, including smooth muscle and neural activities. In mouse (m), estrogen up-regulates the transcript levels of its pore-forming alpha-subunit (Slo, KCNMA1), yet the underlying genomic mechanism(s) is (are) unknown. We first mapped the promoters and regulatory motifs within the mSlo 5'-flanking sequence to subsequently identify genomic regions and mechanisms required for estrogen regulation. mSlo gene has at least two TATA-less promoters with distinct potencies that may direct mSlo transcription from multiple transcription start sites. These qualities mark mSlo as a prototype gene with promoter plasticity capable of generating multiple mRNAs and the potential to adapt to organismal needs. mSlo promoters contain multiple estrogen-responsive sequences, e.g. two quasi-perfect estrogen-responsive elements, ERE1 and ERE2, and Sp1 sites. Accordingly, mSlo promoter activity was highly enhanced by estrogen and blocked by estrogen antagonist ICI 182,780. When promoters are embedded in a 4.91-kb backbone, estrogen responsiveness involves a classical genomic mechanism, via ERE1 and ERE2, that may be complemented by Sp factors, particularly Sp1. Simultaneous but not individual ERE1 and ERE2 mutations caused significant loss of estrogen action. ERE2, which is closer to the proximal promoter, up-regulates this promoter via a classical genomic mechanism. ERE2 strategic position together with ERE1 and ERE2 independence and Sp contribution should ensure mSlo estrogen responsiveness. Thus, the mSlo gene seems to have uniquely evolved to warrant estrogen regulation. Estrogen-mediated mSlo genomic regulation has important implications on long term estrogenic effects affecting smooth muscle and neural functions.

MaxiK channel partners: physiological impact.

The basic functional unit of the large-conductance, voltage- and Ca2+-activated K+ (MaxiK, BK, BKCa) channel is a tetramer of the pore-forming alpha-subunit (MaxiKalpha) encoded by a single gene, Slo, holding multiple alternative exons. Depending on the tissue, MaxiKalpha can associate with modulatory beta-subunits (beta1-beta4) increasing its functional diversity. As MaxiK senses and regulates membrane voltage and intracellular Ca2+, it links cell excitability with cell signalling and metabolism. Thus, MaxiK is a key regulator of vital body functions, like blood flow, uresis, immunity and neurotransmission. Epilepsy with paroxysmal dyskinesia syndrome has been recognized as a MaxiKalpha-related disorder caused by a gain-of-function C-terminus mutation. This channel region is also emerging as a key recognition module containing sequences for MaxiKalpha interaction with its surrounding signalling partners, and its targeting to cell-specific microdomains. The growing list of interacting proteins highlights the possibility that associations with the C-terminus of MaxiKalpha are dynamic and depending on each cellular environment. We speculate that the molecular multiplicity of the C-terminus (and intracellular loops) dictated by alternative exons may modulate or create additional interacting sites in a tissue-specific manner. A challenge is the dissection of MaxiK macromolecular signalling complexes in different tissues and their temporal association/dissociation according to the stimulus.

Molecular and functional signature of heart hypertrophy during pregnancy.

During pregnancy, the heart develops a reversible physiological hypertrophic growth in response to mechanical stress and increased cardiac output; however, underlying molecular mechanisms remain unknown. Here, we investigated pregnancy-related changes in heart structure, function, and gene expression of known markers of pathological hypertrophy and cell stretching in mice hearts. In late pregnancy, hearts show eccentric hypertrophy, as expected for a response to volume overload, with normal left ventricular diastolic function and a moderate reduction in systolic function. Pregnancy-related physiological heart hypertrophy does not induce expression changes of known markers of pathological hypertrophy like: alpha- and beta-myosin heavy chain, atrial natriuretic factor, phospholamban, and sarcoplasmic reticulum Ca2+-ATPase. Instead, it induces the remodeling of Kv4.3 channel and increased c-Src tyrosine kinase activity, a stretch-responsive kinase. Cardiac Kv4.3 channel gene expression was downregulated by approximately 3- to 5-fold, both at the mRNA and protein levels, and was paralleled by a reduction in transient outward K+ currents, a longer action potential and by prolongation of the QT interval. Downregulation of cardiac Kv4.3 transcripts was mimicked by estrogen treatment in ovariectomized mice, and was prevented by the estrogen receptor antagonist ICI 182,780. c-Src activity increased by approximately 2-fold in late pregnancy and after estrogen treatment. We propose that, in addition to mechanical stress, the rise of estrogen toward the end of pregnancy contributes to pregnancy-related heart hypertrophy by increased c-Src activity and that the rise of estrogen is one factor that down regulates cardiac Kv4.3 gene expression providing a molecular correlate for a longer QT interval in pregnancy.

Beta1-subunit of MaxiK channel in smooth muscle: a key molecule which tunes muscle mechanical activity.

The MaxiK channel is the large-conductance, voltage-dependent, and Ca(2+)-activated K(+) channel. This channel is almost ubiquitously distributed among mammalian tissues including smooth muscles. The ability of MaxiK to work as a rheostat fine tuning membrane potential and intracellular Ca(2+) enables it to mediate opposite functions: it facilitates contraction, but also acts as a negative feedback mechanism to restore tone after a contraction cycle. MaxiK activation mediates relaxations to a variety of physiological substances, whereas its inhibition plays a significant role in contractile responses. At the molecular level, MaxiK is a protein complex formed by at least two integral dissimilar membrane subunits, the pore-forming alpha-subunit and a regulatory beta-subunit. In smooth muscles, beta1 is the predominant subunit and most MaxiK seem to be assembled of alpha- and beta1-subunits. The presence of the beta1-subunit confers MaxiK with higher Ca(2+)/voltage sensitivity, which makes this channel an efficient tuner of smooth muscle functions in physiological conditions. The enhanced smooth muscle mechanical activities in mice lacking the beta1-subunit gene support the principal role of this channel molecular component in tissue and whole animal functions. In this review, we discuss MaxiK channel roles as a tuner of smooth muscle contractility, especially focusing attention on the modulatory beta1-subunit.

Coupling of c-Src to large conductance voltage- and Ca2+-activated K+ channels as a new mechanism of agonist-induced vasoconstriction.

The voltage-dependent and Ca(2+)-activated K(+) channel (MaxiK, BK) and the cellular proto-oncogene pp60(c-Src) (c-Src) are abundant proteins in vascular smooth muscle. The role of MaxiK channels as a vasorelaxing force is well established, but their role in vasoconstriction is unclear. Because Src participates in regulating vasoconstriction, we investigated whether c-Src inhibits MaxiK as a mechanism for agonist-induced vasoconstriction. Functional experiments in human and rat show that inhibitors of Src (Lavendustin A, PP2) but not inactive compounds (Lavendustin B, PP3) induce a pronounced relaxation of coronary or aortic smooth muscle precontracted with 5-hydroxytriptamine, phenylephrine, or Angiotensin II. Iberiotoxin, a MaxiK blocker, antagonizes the relaxation induced by Lavendustin A or PP2, indicating that c-Src inhibits the Iberiotoxin-sensitive component, likely MaxiK channels. In agreement, coronary muscle MaxiK currents were enhanced by Lavendustin A. To investigate the molecular mechanism of c-Src action on MaxiK channels, we transiently expressed its alpha subunit, hSlo, with or without c-Src in HEK293T cells. The voltage sensitivity of hSlo was right-shifted by approximately 16 mV. hSlo inhibition by c-Src is due to channel direct phosphorylation because: (i) excised patches exposed to protein tyrosine phosphatase (CD45) resulted in a partial reversal of the inhibitory effect by approximately 10 mV, and (ii) immunoprecipitated hSlo channels were recognized by an anti-phosphotyrosine Ab. Furthermore, coexpression of hSlo and c-Src demonstrate a striking colocalization in HEK293T cells. We propose that MaxiK channels via direct c-Src-dependent phosphorylation play a significant role supporting vasoconstriction after activation of G protein-coupled receptors by vasoactive substances and neurotransmitters.