Xiao-Bo Zhou

Elevated Blood Pressure Linked to Primary Hyperaldosteronism and Impaired Vasodilation in BK Channel–Deficient Mice

Background—Abnormally elevated blood pressure is the most prevalent risk factor for cardiovascular disease. The large-conductance, voltage- and Ca2+-dependent K+ (BK) channel has been proposed as an important effector in the control of vascular tone by linking membrane depolarization and local increases in cytosolic Ca2+ to hyperpolarizing K+ outward currents. However, the BK channel may also affect blood pressure by regulating salt and fluid homeostasis, particularly by adjusting the renin-angiotensin-aldosterone system. Methods and Results—Here we report that deletion of the pore-forming BK channel &agr; subunit leads to a significant blood pressure elevation resulting from hyperaldosteronism accompanied by decreased serum K+ levels as well as increased vascular tone in small arteries. In smooth muscle from small arteries, deletion of the BK channel leads to a depolarized membrane potential, a complete lack of membrane hyperpolarizing spontaneous K+ outward currents, and an attenuated cGMP vasorelaxation associated with a reduced suppression of Ca2+ transients by cGMP. The high level of BK channel expression observed in wild-type adrenal glomerulosa cells, together with unaltered serum renin activities and corticotropin levels in mutant mice, suggests that the hyperaldosteronism results from abnormal adrenal cortical function in BK−/− mice. Conclusions—These results identify previously unknown roles of BK channels in blood pressure regulation and raise the possibility that BK channel dysfunction may underlie specific forms of hyperaldosteronism.

Protein Phosphatase 2A Is Essential for the Activation of Ca2+-activated K+ Currents by cGMP-dependent Protein Kinase in Tracheal Smooth Muscle and Chinese Hamster Ovary Cells

The regulation of Ca2+-activated K+ channels (KCa channels) by cGMP-dependent protein kinase (cGMP kinase) and its molecular mechanism were investigated in Chinese hamster ovary (CHO) and tracheal smooth muscle cells. In CHO wild-type cells (CHO-WT cells) and in CHO cells stably transfected with cGMP kinase Iα (CHO-cGK cells), KCa channels with intermediate conductance (∼50 picosiemens) were identified. Due to the basal activity of cGMP kinase, Ca2+-activated K+ currents had a higher sensitivity toward the cytosolic Ca2+ concentration in CHO-cGK cells than in CHO-WT cells. Dialysis of the active fragment of cGMP kinase (300 nM) into CHO-WT cells or of cGMP into CHO-cGK cells increased the Ca2+-activated K+ current, while the catalytic subunit of cAMP-dependent protein kinase (cAMP kinase) was without effect. In cell-attached patches obtained from freshly isolated bovine tracheal smooth muscle cells, the open state probability (NPo) of maxi-KCa channels (conductance of ∼260 picosiemens) was enhanced by 300 μM 8-(4-chlorophenylthio)-cGMP, a specific and potent activator of cGMP kinase. In contrast, 1 μM isoprenaline, 20 μM forskolin, and 3 mM 8-bromo-cAMP failed to enhance KCa channel activity. In excised inside-out patches, only the active fragment of cGMP kinase (but not that of cAMP kinase) increased NPo when applied to the cytosolic side of the patch. The enhancement of NPo by cGMP kinase was inhibited in CHO cells as well as in tracheal smooth muscle cells by the cGMP kinase inhibitor KT 5823 (1 μM) and the protein phosphatase (PP) inhibitors microcystin (5 μM) and okadaic acid (10 nM). The catalytic subunit of PP2A (but not that of PP1) mimicked the effect of cGMP kinase on NPo in excised inside-out patches. The results show that cGMP kinase regulates two different KCa channels in two unrelated cell types by the same indirect mechanism, which requires the activity of PP2A. The regulation of the KCa channel is specific for cGMP kinase and is not mimicked by cAMP kinase.

A Molecular Switch for Specific Stimulation of the BKCa Channel by cGMP and cAMP Kinase

The cGMP and the cAMP pathways control smooth muscle tone by regulation of BKCa (BK) channel activity. BK channels show considerable diversity and plasticity in their regulation by cyclic nucleotide-dependent protein kinases. The underlying molecular mechanisms are unclear but may involve expression of splice variants of the BK channel α subunit. Three isoforms, BKA, BKB, and BKC, which were cloned from tracheal smooth muscle, differed only in their C terminus. When expressed in HEK293 cells, cGMP kinase (cGK) but not cAMP kinase (cAK) stimulated the activity of BKA and BKB by shifting the voltage dependence of the channel to more negative potentials. In contrast, BKC was exclusively stimulated by cAK. BKC lacks a C-terminal tandem phosphorylation motif for protein kinase C (PKC) with Ser1151 and Ser1154. Mutation of this motif in BKA switched channel regulation from cGK to cAK. Furthermore, inhibition of PKC in excised patches from cells expressing BKA abolished the stimulatory effect of cGK but allowed channel stimulation by cAK. cAK and cGK phosphorylated the channel at different sites. Thus, phosphorylation/dephosphorylation by PKC determines whether the BK channel is stimulated by cGK or cAK. The molecular mechanisms may be relevant for smooth muscle relaxation by cAMP and cGMP.

Role of small-conductance calcium-activated potassium channels in atrial electrophysiology and fibrillation in the dog.

Background— Recent evidence points to functional Ca2+-dependent K+ (SK) channels in the heart that may govern atrial fibrillation (AF) risk, but the underlying mechanisms are unclear. This study addressed the role of SK channels in atrial repolarization and AF persistence in a canine AF model. Methods and Results— Electrophysiological variables were assessed in dogs subjected to atrial remodeling by 7-day atrial tachypacing (AT-P), as well as controls. Ionic currents and single-channel properties were measured in isolated canine atrial cardiomyocytes by patch clamp. NS8593, a putative selective SK blocker, suppressed SK current with an IC50 of ≈5 &mgr;mol/L, without affecting Na+, Ca2+, or other K+ currents. Whole-cell SK current sensitive to NS8593 was significantly larger in pulmonary vein (PV) versus left atrial (LA) cells, without a difference in SK single-channel open probability (Po), whereas AT-P enhanced both whole-cell SK currents and single-channel Po. SK-current block increased action potential duration in both PV and LA cells after AT-P; but only in PV cells in absence of AT-P. SK2 expression was more abundant at both mRNA and protein levels for PV versus LA in control dogs, in both control and AT-P; AT-P upregulated only SK1 at the protein level. Intravenous administration of NS8593 (5 mg/kg) significantly prolonged atrial refractoriness and reduced AF duration without affecting the Wenckebach cycle length, left ventricular refractoriness, or blood pressure. Conclusions— SK currents play a role in canine atrial repolarization, are larger in PVs than LA, are enhanced by atrial-tachycardia remodeling, and appear to participate in promoting AF maintenance. These results are relevant to the potential mechanisms underlying the association between SK single-nucleotide polymorphisms and AF and suggest SK blockers as potentially interesting anti-AF drugs.

Dual role of protein kinase C on BK channel regulation

Large conductance voltage- and Ca2+-activated potassium channels (BK channels) are important feedback regulators in excitable cells and are potently regulated by protein kinases. The present study reveals a dual role of protein kinase C (PKC) on BK channel regulation. Phosphorylation of S695 by PKC, located between the two regulators of K+ conductance (RCK1/2) domains, inhibits BK channel open-state probability. This PKC-dependent inhibition depends on a preceding phosphorylation of S1151 in the C terminus of the channel α-subunit. Phosphorylation of only one α-subunit at S1151 and S695 within the tetrameric pore is sufficient to inhibit BK channel activity. We further detected that protein phosphatase 1 is associated with the channel, constantly counteracting phosphorylation of S695. PKC phosphorylation at S1151 also influences stimulation of BK channel activity by protein kinase G (PKG) and protein kinase A (PKA). Though the S1151A mutant channel is activated by PKA only, the phosphorylation of S1151 by PKC renders the channel responsive to activation by PKG but prevents activation by PKA. Phosphorylation of S695 by PKC or introducing a phosphomimetic aspartate at this position (S695D) renders BK channels insensitive to the stimulatory effect of PKG or PKA. Therefore, our findings suggest a very dynamic regulation of the channel by the local PKC activity. It is shown that this complex regulation is not only effective in recombinant channels but also in native BK channels from tracheal smooth muscle.

Upregulation of K(2P)3.1 K+ Current Causes Action Potential Shortening in Patients With Chronic Atrial Fibrillation

Background— Antiarrhythmic management of atrial fibrillation (AF) remains a major clinical challenge. Mechanism-based approaches to AF therapy are sought to increase effectiveness and to provide individualized patient care. K2P3.1 (TASK-1 [tandem of P domains in a weak inward-rectifying K+ channel–related acid-sensitive K+ channel-1]) 2-pore-domain K+ (K2P) channels have been implicated in action potential regulation in animal models. However, their role in the pathophysiology and treatment of paroxysmal and chronic patients with AF is unknown. Methods and Results— Right and left atrial tissue was obtained from patients with paroxysmal or chronic AF and from control subjects in sinus rhythm. Ion channel expression was analyzed by quantitative real-time polymerase chain reaction and Western blot. Membrane currents and action potentials were recorded using voltage- and current-clamp techniques. K2P3.1 subunits exhibited predominantly atrial expression, and atrial K2P3.1 transcript levels were highest among functional K2P channels. K2P3.1 mRNA and protein levels were increased in chronic AF. Enhancement of corresponding currents in the right atrium resulted in shortened action potential duration at 90% of repolarization (APD90) compared with patients in sinus rhythm. In contrast, K2P3.1 expression was not significantly affected in subjects with paroxysmal AF. Pharmacological K2P3.1 inhibition prolonged APD90 in atrial myocytes from patients with chronic AF to values observed among control subjects in sinus rhythm. Conclusions— Enhancement of atrium-selective K2P3.1 currents contributes to APD shortening in patients with chronic AF, and K2P3.1 channel inhibition reverses AF-related APD shortening. These results highlight the potential of K2P3.1 as a novel drug target for mechanism-based AF therapy.

REGULATION OF STABLY EXPRESSED AND NATIVE BK CHANNELS FROM HUMAN MYOMETRIUM BY CGMP- AND CAMP-DEPENDENT PROTEIN KINASE

Abstract The cloned BK channel α subunit from human myometrium was stably expressed in Chinese hamster ovary cells, either alone (CHOα cells) or in combination with the auxiliary β subunit (CHOα+β cells). We studied basic channel properties and the effects of cGMP- and cAMP-dependent protein kinases on the BK channel activity. Coexpression of α and β subunits enhanced the Ca2+ and voltage sensitivity of the BK channel, and decreased the inhibitory potency of iberiotoxin. Blocking and stimulating effects on BK channel activity by charybdotoxin and nitric oxide, respectively, were independent of the β subunit. The cGMP kinase Iα and cAMP kinase failed to affect BK channel activity in CHOα and CHOα+β cells at different [Ca2+]i and voltages. In contrast, BK channels in freshly isolated myometrial cells from postmenopausal women responded to cAMP kinase and cGMP kinase with a fourfold and twofold decrease in their open probability (NPo), respectively. These effects could be reversed by alkaline phosphatase and remained unaffected by the phosphatase inhibitor okadaic acid (100 nM). In 28% of myometrial cells, however, cAMP and cGMP kinases increased NPo 2-fold and 3.5-fold, respectively. This stimulation was enhanced rather than reversed by alkaline phosphatase and was abolished by 100 nM okadaic acid. The results suggest that in stably transfected CHO cells the expressed BK channel is not regulated by cAMP kinase and cGMP kinase. However, in native myometrial cells stimulatory and inhibitory regulation of BK channels by cAMP kinase and cGMP kinase was observed, suggesting that channel regulation by the protein kinases requires factors that are not provided by CHO cells. Alternatively, failure of regulation may have been due to the primary structure of the myometrial BK channel protein used in this study.

Palmitoylation and Membrane Association of the Stress Axis Regulated Insert (STREX) Controls BK Channel Regulation by Protein Kinase C

Background: Large-conductance potassium (BK) channels containing the stress axis regulated insert (STREX) are important regulators of cellular excitability. Results: Membrane detachment of STREX by protein kinase A (PKA)-mediated phosphorylation or de-palmitoylation established channel inhibition by protein kinase C (PKC). Conclusion: Membrane association of STREX regulates the access of PKC to its inhibitory phosphorylation site. Significance: The interplay of phosphorylation by PKA, PKC, and palmitoylation determines BK-STREX channel activity. Large-conductance, calcium- and voltage-gated potassium (BK) channels play an important role in cellular excitability by controlling membrane potential and calcium influx. The stress axis regulated exon (STREX) at splice site 2 inverts BK channel regulation by protein kinase A (PKA) from stimulatory to inhibitory. Here we show that palmitoylation of STREX controls BK channel regulation also by protein kinase C (PKC). In contrast to the 50% decrease of maximal channel activity by PKC in the insertless (ZERO) splice variant, STREX channels were completely resistant to PKC. STREX channel mutants in which Ser700, located between the two regulatory domains of K+ conductance (RCK) immediately downstream of the STREX insert, was replaced by the phosphomimetic amino acid glutamate (S700E) showed a ∼50% decrease in maximal channel activity, whereas the S700A mutant retained its normal activity. BK channel inhibition by PKC, however, was effectively established when the palmitoylation-mediated membrane-anchor of the STREX insert was removed by either pharmacological inhibition of palmitoyl transferases or site-directed mutagenesis. These findings suggest that STREX confers a conformation on BK channels where PKC fails to phosphorylate and to inhibit channel activity. Importantly, PKA which inhibits channel activity by disassembling the STREX insert from the plasma membrane, allows PKC to further suppress the channel gating independent from voltage and calcium. Our results present an important example for the cross-talk between ion channel palmitoylation and phosphorylation in regulation of cellular excitability.

Pregnancy switches adrenergic signal transduction in rat and human uterine myocytes as probed by BKCa channel activity

1 We used large conductance Ca2+‐activated K+ (BKCa) channel activity as a probe to characterize the inhibitory/stimulatory G protein (Gi/Gs) signalling pathways in intact cells from pregnant (PM) and non‐pregnant (NPM) myometrium. 2 Isoprenaline (10 μM) enhanced the outward current (Iout) in PM cells and inhibited Iout in NPM cells. Additional application of the α2‐adrenoceptor (α2‐AR) agonist clonidine (10 μM) further enhanced the isoprenaline‐modulated Iout in PM cells but partially antagonized Iout in NPM cells. Clonidine alone did not affect Iout. The specific cAMP kinase (PKA) inhibitor H‐89 (1 μM) abolished the effects of isoprenaline and clonidine. The specific BKCa channel blocker iberiotoxin (0·1 μM) inhibited Iout by ≈80 %; the residual current was insensitive to isoprenaline. 3 Inhibition of Gi activity by either pertussis toxin or the GTPase activating protein RGS16 abolished inhibitory as well as stimulatory effects of clonidine on Iout. 4 Transducin‐α, a scavenger of Giβγ dimers, converted the stimulatory action of clonidine on Iout into an inhibitory effect. Free transducin‐βγ enhanced both the stimulatory and the inhibitory effects of isoprenaline on Iout. 5 The results demonstrate that BKCa channel activity is a sensitive probe to follow adenylyl cyclase‐cAMP‐PKA signalling in myometrial smooth muscle cells. Both Giα‐mediated inhibition and Giβγ‐mediated stimulation can occur in the same cell, irrespective of pregnancy. It is speculated that the coupling between α2‐AR and Gi proteins is more efficient during pregnancy and that Giβγ at high levels simply override the inhibitory action of Giα.

Reduced rather than enhanced cholinergic airway constriction in mice with ablation of the large conductance Ca2+-activated K+ channel

The unique voltage‐ and Ca2+‐depen‐dent K+ (BK) channel, prominently expressed in airway smooth muscle cells, has been suggested as an important effector in controlling airway contractility. Its deletion in mice depolarized resting membrane potential of tracheal cells, suggesting an increased open‐probability of voltage‐gated Ca2+ channels. While car‐bachol concentration‐dependently increased the tonic tension of wild‐type (WT) trachea, mutant trachea showed a different response with rapid tension devel‐opment followed by phasic contractions superimposed on a tonic component. Tonic contractions were sub‐stantially more dependent on L‐type Ca2+ current in mutant than in WT trachea, even though L‐type Ca2+ channels were not up‐regulated. In the absence of L‐type Ca2+ current, half‐maximal contraction of trachea was shifted from 0.51 to 1.7 μM. In agreement, cholinergic bronchoconstriction was reduced in mutant lung slices, isolated‐perfused lungs and, most impressively, in mutant mice analyzed by body plethysmography. Furthermore, isoprenaline‐mediated airway relaxation was enhanced in mutants. In‐depfh analysis of cAMP and cGMP signaling revealed up‐regulation of the cGMP pathway in mutant tracheal muscle. Inhibition of cGMP kinase reestablished normal sensitivity toward carbachol, indicating that up‐regulation of cGMP signaling counterbalances for BK channel ablation, pointing to a predominant role of BK channel in regulation of airway tone.—Sausbier, M., Zhou, X.‐B., Beier, C., Sausbier, U., Wolpers, D., Maget, S., Martin, C., Dietrich, A., Ressmeyer, A.‐R., Renz, H., Schlossmann, J., Hofmann, F., Neuhuber, W., Gudermann, T., Uhlig, S., Korth, M., Ruth, P. Reduced rather than enhanced cholinergic airway constriction in mice with ablation of the large conductance Ca2+‐activated K+ channel. FASEB J. 21, 812–822 (2007)