The basic functional unit of the large-conductance, voltage-and Ca 2+ -activated K + (MaxiK, BK, BK Ca ) 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 Ca 2+ , 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.

Abstract-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: ␣-and ␤-myosin heavy chain, atrial natriuretic factor, phospholamban, and sarcoplasmic reticulum Ca 2ϩ -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 Ϸ3-to 5-fold, both at the mRNA and protein levels, and was paralleled by a reduction in transient outward K ϩ 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 Ϸ2-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. Key Words: heart hypertrophy Ⅲ pregnancy Ⅲ estrogen Ⅲ I to Ⅲ Kv4.3 channel D uring pregnancy, the heart undergoes hypertrophic growth to compensate for the increased cardiac output. Cardiac hypertrophy has been defined as an increase in cardiomyocyte size that can be beneficial and adaptive (physiological) or a maladaptive (pathophysiological) phenomenon to compensate for the hemodynamic stress resulting from pressure or volume overloads. Pressure overload induces concentric hypertrophy characterized by wall thickening without significant chamber enlargement. Volume overload, as in pregnancy, induces eccentric hypertrophy characterized by chamber enlargement with a proportional change in wall thickness. 1 Physiological hypertrophy is reversible and occurs during maturation, pregnancy, and exercise without morbid effects on cardiac function. [2][3][4] Despite the growing knowledge of the molecular changes that can occur during pathological heart hypertrophy, 1 the underlying molecular mechanisms of pregnancy-related heart hypertrophy are unknown.In pathological heart hypertrophy, expression of a set of genes has been reported to be altered and, therefore, can be used as markers for this class of hypertrophy. Examples are, ␣-, ␤-myosin heavy c...

(2), or as a negative feedback mechanism to limit Ca 2ϩ entry by hyperpolarizing the plasma membrane and closing voltagedependent Ca 2ϩ channels (3) previously opened by pressure (4) or vasoconstrictors like 5-hydroxytriptamine (5-HT) and Angiotensin II (AngII) (5, 6). In vitro evidence suggests that MaxiK also may play a role in vasoconstriction as it is inhibited by the potent constrictors AngII (7) and thromboxane A2 (8) in bilayers. However, the functional role of MaxiK channels in agonist-induced contraction has not been demonstrated.Pharmacomechanical and biochemical evidence indicate that one mechanism of agonist-induced contraction may involve tyrosine phosphorylation͞dephosphorylation with phosphorylation associated with vasoconstriction (9). However, most studies have been performed by using inhibitors with broad actions (e.g., tyrphostin and genistein) (10, 11). Using more selective inhibitors for Src-family tyrosine kinases, PP1 and PP2 (12) recent studies in rat aorta (13) and mesenteric arteries (14) show that 5-HT and AngII contractions involve a Src tyrosine kinase, likely c-Src. However, the downstream effector(s) of c-Src promoting vasoconstriction are unknown.We hypothesized that MaxiK may be a potential downstream effector of c-Src favoring vasoconstriction. This is based on the facts that both c-Src tyrosine kinase and MaxiK are particularly abundant in smooth muscles including the vasculature (15-18), and that Lavendustin A (LavA), a c-Src and Lck inhibitor (10,19), increases the activity of rat tail artery MaxiK (20). Here, we provide evidence showing that agonist-induced vasoconstriction by 5-HT, AngII, and phenylephrine involves inhibition of MaxiK channels by c-Src via direct phosphorylation of the channel protein. This new signaling pathway has a significant role in human and rat vasoconstriction, providing a link between electromechanical and pharmacomechanical coupling (21). Furthermore, the results indicate that MaxiK channels can function as a rheostat controlling both vasoconstriction and vasorelaxation. Experimental ProceduresTissue. Human coronary arteries were obtained from explanted hearts (University of California, Los Angeles, Medical Center). Male 3-mo-old F344 rats were used. Protocols received institutional approval.Isometric Contraction. Arterial rings (2.0-to 3.0-mm internal diameter, 3 mm long) without endothelium were stretched to an optimal resting tension (2 g, human coronaries; 1.2 g, rat aorta) and equilibrated for 60 min in Krebs solution. Percentage relaxation after drug application was calculated for tonic contractions from: percentage relaxation ϭ (max -min)͞(maxbasal) ϫ 100; where max ϭ maximal steady-state tension after constricting agonist stimulation, min ϭ minimal tension attained after drug application, basal ϭ basal tension prior constricting agonist stimulation. For phasic contractions, percentage relaxation ϭ (cycles͞h before drug application ͞cycles͞h after drug application ) ϫ 100. Because IbTx treatment abolished phasic contractions, in this case p...

Native large conductance, voltage-dependent, and Ca 2؉ -sensitive K ؉ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, we used the human poreforming ␣-subunit of the Ca 2؉ -sensitive K ؉ channel, Hslo, and the ␣-isoform of cGMP-dependent protein kinase I. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by ϳ20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates ␥-33 P into the immunopurified Hslo band of ϳ125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the ␣-subunit of large conductance Ca 2؉ -sensitive K ؉ channels is substrate for G-I␣ kinase in vivo and support direct phosphorylation as a mechanism for PKG-I␣-induced activation of maxi-K channels.

We identified a novel MaxiK alpha subunit splice variant (SV1) from rat myometrium that is also present in brain. SV1 has a 33-amino acid insert in the S1 transmembrane domain that does not alter S1 overall hydrophobicity, but makes the S0-S1 linker longer. SV1 was transfected in HEK293T cells and studied using immunocytochemistry and electrophysiology. In non-permeabilized cells, N-terminal c-Myc- or C-terminal green fluorescent protein-tagged SV1 displayed no surface labeling or currents. The lack of SV1 functional expression was due to endoplasmic reticulum (ER) retention as determined by colabeling experiments with a specific ER marker. To explore the functional role of SV1, we coexpressed SV1 with the alpha (human SLO) and beta1 (KCNMB1) subunits of the MaxiK channel. Coexpression of SV1 inhibited surface expression of alpha and beta1 subunits approximately 80% by trapping them in the ER. This inhibition seems to be specific for MaxiK channel subunits since SV1 was unable to prevent surface expression of the Kv4.3 channel or to interact with green fluorescent protein. These results indicate a dominant-negative role of SV1 in MaxiK channel expression. Moreover, they reveal down-regulation by splice variants as a new mechanism that may contribute to the diverse levels of MaxiK channel expression in non-excitable and excitable cells.