Abderrahmane Alioua

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 α‐subunit (MaxiKα) encoded by a single gene, Slo, holding multiple alternative exons. Depending on the tissue, MaxiKα can associate with modulatory β‐subunits (β1–β4) 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 MaxiKα‐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 MaxiKα 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 MaxiKα 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: &agr;- and &bgr;-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 ≈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 ≈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.

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 Ca2+-activated K+ channel (MaxiK, BK) and the cellular proto-oncogene pp60c-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 α subunit, hSlo, with or without c-Src in HEK293T cells. The voltage sensitivity of hSlo was right-shifted by ≈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 ≈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.

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 α-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 β1-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, φXXXXφXXφ. Deletion of 1007YNMLCFGIY1015 caused ∼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.

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

The large conductance, voltage- and Ca2+-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 α-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.

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‐HT2AR 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‐HT2AR‐mediated contraction. Inhibition of Src kinase activity with 10 μm 4‐amino‐5‐(4‐chlorophenyl)‐7‐(t‐butyl)pyrazolo[3,4‐d]pyrimidine (PP2) prior to contraction resulted in ∼90–99% inhibition of contractions induced by 5‐HT or by α‐methyl‐5‐HT (5‐HT2R agonist). In contrast, PP2 pretreatment only partly inhibited contractions induced by angiotensin II and the thromboxane A2 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 IC50 of ∼1 μm. Ketanserin (10 nm), a 5‐HT2A antagonist, but not antagonists of 5‐HT2BR (100 nm SB204741) or 5‐HT2CR (20 nm RS102221), prevented 5‐HT‐induced contractions, mimicking PP2 and implicating 5‐HT2AR as the major receptor subtype coupled to c‐Src. In HEK 293T cells, c‐Src and 5‐HT2AR 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‐HT2AR contractile signalling in aorta.

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, BKCa) are well known for sustaining cerebral and coronary arterial tone and for their linkage to vasodilator β-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 receptors 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.

Ca2+- and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: From microtubules to the plasma membrane

Large‐conductance, voltage‐ and Ca2+‐activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore‐forming α‐subunits (MaxiKα) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show that MaxiKα is predominantly confined to the microtubule network. Distinct microtubule distribution of MaxiKα was visualized by three independent labeling approaches: (1) MaxiKα‐specific antibodies, (2) expressed EGFP‐labeled MaxiKα, and (3) fluorophore‐conjugated iberiotoxin, a specific MaxiK pore‐blocker. This MaxiKα association with microtubules was further confirmed by in vitro His‐tag pulldown, co‐immunoprecipitation from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine or thapsigargin, resulted in increased MaxiKα labeling at the plasma membrane. More notably, U46619, an analog of thromboxane A2 (TXA2), which triggers Ca2+‐release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiKα surface labeling. Whole‐cell patch clamp recordings of U46619‐stimulated cells develop a ∼3‐fold increase in current amplitude indicating that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. While microtubules are largely absent in mature astrocytes, immunohistochemistry results in brain slices show that cortical astrocytes in the newborn mouse (P1) exhibit a robust expression of microtubules that significantly colocalize with MaxiKα. The results of this study provide the novel insight that suggests that Ca2+ released from intracellular stores may play a key role in regulating the traffic of intracellular, microtubule‐associated MaxiKα stores to the plasma membrane of developing murine astrocytes.

Unconventional myristoylation of large-conductance Ca2+-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 Ca2+-activated K+ channel (hSlo1) might undergo internal myristoylation. hSlo1 expressed in HEK293T cells incorporated [3H]myristic acid via a posttranslational mechanism, which is insensitive to cycloheximide, an inhibitor of protein biosynthesis. In-gel hydrolysis of [3H]myristoyl-hSlo1 with alkaline NH2OH (which cleaves hydroxyesters) but not neutral NH2OH (which cleaves thioesters) completely removed [3H]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.

The first transmembrane domain (TM1) of β2‐subunit binds to the transmembrane domain S1 of α‐subunit in BK potassium channels

BK channel subunit alpha physically interacts with BK channel subunit beta‐2 by anti tag coimmunoprecipitation(View interaction)