Critical role for cochlear hair cell BK channels for coding the temporal structure and dynamic range of auditory information for central auditory processing.

Large conductance, voltage- and Ca(2+)-activated K(+) (BK) channels in inner hair cells (IHCs) of the cochlea are essential for hearing. However, germline deletion of BKα, the pore-forming subunit KCNMA1 of the BK channel, surprisingly did not affect hearing thresholds in the first postnatal weeks, even though altered IHC membrane time constants, decreased IHC receptor potential alternating current/direct current ratio, and impaired spike timing of auditory fibers were reported in these mice. To investigate the role of IHC BK channels for central auditory processing, we generated a conditional mouse model with hair cell-specific deletion of BKα from postnatal day 10 onward. This had an unexpected effect on temporal coding in the central auditory system: neuronal single and multiunit responses in the inferior colliculus showed higher excitability and greater precision of temporal coding that may be linked to the improved discrimination of temporally modulated sounds observed in behavioral training. The higher precision of temporal coding, however, was restricted to slower modulations of sound and reduced stimulus-driven activity. This suggests a diminished dynamic range of stimulus coding that is expected to impair signal detection in noise. Thus, BK channels in IHCs are crucial for central coding of the temporal fine structure of sound and for detection of signals in a noisy environment.

Osteopenia due to enhanced cathepsin K release by BK channel ablation in osteoclasts.

BACKGROUND: The process of bone resorption by osteoclasts is regulated by Cathepsin K, the lysosomal collagenase responsible for the degradation of the organic bone matrix during bone remodeling. Recently, Cathepsin K was regarded as a potential target for therapeutic intervention of osteoporosis. However, mechanisms leading to osteopenia, which is much more common in young female population and often appears to be the clinical pre-stage of idiopathic osteoporosis, still remain to be elucidated, and molecular targets need to be identified. METHODOLOGY/PRINCIPAL FINDINGS: We found, that in juvenile bone the large conductance, voltage and Ca(2+)-activated (BK) K(+) channel, which links membrane depolarization and local increases in cytosolic calcium to hyperpolarizing K(+) outward currents, is exclusively expressed in osteoclasts. In juvenile BK-deficient (BK(-/-)) female mice, plasma Cathepsin K levels were elevated two-fold when compared to wild-type littermates. This increase was linked to an osteopenic phenotype with reduced bone mineral density in long bones and enhanced porosity of trabecular meshwork in BK(-/-) vertebrae as demonstrated by high-resolution flat-panel volume computed tomography and micro-CT. However, plasma levels of sRANKL, osteoprotegerin, estrogene, Ca(2+) and triiodthyronine as well as osteoclastogenesis were not altered in BK(-/-) females. CONCLUSION/SIGNIFICANCE: Our findings suggest that the BK channel controls resorptive osteoclast activity by regulating Cathepsin K release. Targeted deletion of BK channel in mice resulted in an osteoclast-autonomous osteopenia, becoming apparent in juvenile females. Thus, the BK(-/-) mouse-line represents a new model for juvenile osteopenia, and revealed the BK channel as putative new target for therapeutic controlling of osteoclast activity.

Functional significance of the intermediate conductance Ca2+-activated K+ channel for the short-term survival of injured erythrocytes.

Increased cytosolic Ca(2+) concentrations activate Gardos K(+) channels in human erythrocytes with membrane hyperpolarization, efflux of K(+), Cl⁻, and osmotically obliged H2O resulting in cell shrinkage, a phenomenon referred to as Gardos effect. We tested whether the Gardos effect delays colloid osmotic hemolysis of injured erythrocytes from mice lacking the Ca(2+)-activated K(+) channel K(Ca)3.1. To this end, we applied patch clamp and flow cytometry and determined in vitro as well as in vivo hemolysis. As a result, erythrocytes from K(Ca)3.1-deficient (K(Ca)3.1(-/-)) mice lacked Gardos channel activity and the Gardos effect. Blood parameters, reticulocyte count, or osmotic erythrocyte resistance, however, did not differ between K(Ca)3.1(-/-) mice and their wild-type littermates, suggesting low or absent Gardos channel activity in unstressed erythrocytes. Oxidative stress-induced Ca(2+) entry and phospholipid scrambling were significantly less pronounced in K(Ca)3.1(-/-) than in wild-type erythrocytes. Moreover, in vitro treatment with α-toxin from Staphylococcus aureus, which forms pores in the cellular membrane, resulted in significantly stronger hemolysis of K(Ca)3.1(-/-) than of wild-type erythrocytes. Intravenous injection of α-toxin induced more profound hemolysis in K(Ca)3.1(-/-) than in wild-type mice. Similarly, intra-peritoneal application of the redox-active substance phenylhydrazine, an agent for the induction of hemolytic anemia, was followed by a significantly stronger decrease of hematocrit in K(Ca)3.1(-/-) than in wild-type mice. Finally, malaria infection triggered the activation of K(Ca)3.1 and transient shrinkage of the infected erythrocytes. In conclusion, K(Ca)3.1 channel activity and Gardos effect counteract hemolysis of injured erythrocytes, thus decreasing hemoglobin release into circulating blood.

Dual role of protein kinase C on BK channel regulation.

Large conductance voltage- and Ca(2+)-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 S(695) 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 S(1151) in the C terminus of the channel alpha-subunit. Phosphorylation of only one alpha-subunit at S(1151) and S(695) 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 S(695). PKC phosphorylation at S(1151) also influences stimulation of BK channel activity by protein kinase G (PKG) and protein kinase A (PKA). Though the S(1151)A mutant channel is activated by PKA only, the phosphorylation of S(1151) by PKC renders the channel responsive to activation by PKG but prevents activation by PKA. Phosphorylation of S(695) by PKC or introducing a phosphomimetic aspartate at this position (S(695)D) 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.

BK channels control cerebellar Purkinje and Golgi cell rhythmicity in vivo.

Calcium signaling plays a central role in normal CNS functioning and dysfunction. As cerebellar Purkinje cells express the major regulatory elements of calcium control and represent the sole integrative output of the cerebellar cortex, changes in neural activity- and calcium-mediated membrane properties of these cells are expected to provide important insights into both intrinsic and network physiology of the cerebellum. We studied the electrophysiological behavior of Purkinje cells in genetically engineered alert mice that do not express BK calcium-activated potassium channels and in wild-type mice with pharmacological BK inactivation. We confirmed BK expression in Purkinje cells and also demonstrated it in Golgi cells. We demonstrated that either genetic or pharmacological BK inactivation leads to ataxia and to the emergence of a beta oscillatory field potential in the cerebellar cortex. This oscillation is correlated with enhanced rhythmicity and synchronicity of both Purkinje and Golgi cells. We hypothesize that the temporal coding modification of the spike firing of both Purkinje and Golgi cells leads to the pharmacologically or genetically induced ataxia.

Heme oxygenase-2 and large-conductance Ca2+-activated K+ channels: lung vascular effects of hypoxia.

RATIONALE: Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which pulmonary gas exchange is optimized by the adaptation of blood flow to alveolar ventilation. In chronic hypoxia, in addition to HPV a vascular remodeling process leads to pulmonary hypertension. A complex of heme oxygenase-2 (HO-2) and the BK channel has been suggested as a universal oxygen sensor system. OBJECTIVES: We investigated whether this complex serves as an oxygen sensor for the vascular effects of alveolar hypoxia in the lung. METHODS: The investigations were performed in chronically hypoxic mice, in isolated perfused and ventilated lungs, and on the cellular level, including HO-2- and BK-channel deficient mice. MEASUREMENTS AND MAIN RESULTS: Immunohistochemical analysis of mouse lungs identified HO-2 mainly in pulmonary arteries, the bronchial epithelium, and alveolar epithelial cells. BK channel alpha-subunit (BKalpha) immunoreactivity was found primarily in the bronchial and vascular smooth muscle layer. Immunofluorescence staining and coimmunoprecipitation suggested only a weak complexation of HO-2 and BKalpha in pulmonary arterial smooth muscle cells. The strength of acute and sustained HPV, determined in isolated perfused and ventilated lungs, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice. Exposure of mice to 3 weeks of chronic hypoxia resulted in a slight down-regulation of HO-2 and no alteration in BKalpha expression. The degree of pulmonary hypertension that developed, quantified on the basis of right ventricular pressure, right-heart hypertrophy, and the degree of muscularization of precapillary pulmonary arteries, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice. CONCLUSIONS: It is demonstrated that neither deletion of HO-2 nor BK channels affect acute, sustained, and chronic vascular responses to alveolar hypoxia in the lung.

Inducible knockout mutagenesis reveals compensatory mechanisms elicited by constitutive BK channel deficiency in overactive murine bladder.

The large-conductance, voltage-dependent and Ca(2+)-dependent K(+) (BK) channel links membrane depolarization and local increases in cytosolic free Ca(2+) to hyperpolarizing K(+) outward currents, thereby controlling smooth muscle contractility. Constitutive deletion of the BK channel in mice (BK(-/-)) leads to an overactive bladder associated with increased intravesical pressure and frequent micturition, which has been revealed to be a result of detrusor muscle hyperexcitability. Interestingly, time-dependent and smooth muscle-specific deletion of the BK channel (SM-BK(-/-)) caused a more severe phenotype than displayed by constitutive BK(-/-) mice, suggesting that compensatory pathways are active in the latter. In detrusor muscle of BK(-/-) but not SM-BK(-/-) mice, we found reduced L-type Ca(2+) current density and increased expression of cAMP kinase (protein kinase A; PKA), as compared with control mice. Increased expression of PKA in BK(-/-) mice was accompanied by enhanced beta-adrenoceptor/cAMP-mediated suppression of contractions by isoproterenol. This effect was attenuated by about 60-70% in SM-BK(-/-) mice. However, the Rp isomer of adenosine-3',5'-cyclic monophosphorothioate, a blocker of PKA, only partially inhibited enhanced cAMP signaling in BK(-/-) detrusor muscle, suggesting the existence of additional compensatory pathways. To this end, proteome analysis of BK(-/-) urinary bladder tissue was performed, and revealed additional compensatory regulated proteins. Thus, constitutive and inducible deletion of BK channel activity unmasks compensatory mechanisms that are relevant for urinary bladder relaxation.

Aldosterone increases KCa1.1 (BK) channel-mediated colonic K+ secretion.

Mammalian K(+) homeostasis results from highly regulated renal and intestinal absorption and secretion, which balances the unregulated K(+) intake. Aldosterone is known to enhance both renal and colonic K(+) secretion. In mouse distal colon K(+) secretion occurs exclusively via luminal K(Ca)1.1 (BK) channels. Here we investigate if aldosterone stimulates colonic K(+) secretion via BK channels. Luminal Ba(2+) and iberiotoxin (IBTX)-sensitive electrogenic K(+) secretion was measured in Ussing chambers. In vivo aldosterone was augmented via a high K(+) diet. High K(+) diet led to a 2-fold increase of luminal Ba(2+) and IBTX-sensitive short-circuit current in distal mouse colonic mucosa. This effect was absent in BK alpha-subunit-deficient (BK(-/-)) mice. The resting and diet-induced K(+) secretion was stimulated by luminal ionomycin. In BK(-/-) mice luminal ionomycin did not stimulate K(+) secretion. In vitro addition of aldosterone likewise triggered a 2-fold increase in K(+) secretion, which was inhibited by the mineralocorticoid receptor antagonist spironolactone and the BK channel blocker IBTX. Semi-quantification of mRNA from colonic crypts showed up-regulation of BK alpha- and beta(2)-subunits in high K(+) diet mice. The BK channel could be detected luminally in colonic crypt cells by immunohistochemistry. The expression level of the channel in the luminal membrane was strongly up-regulated in K(+)-loaded animals. Taken together, these data strongly suggest that aldosterone-induced K(+) secretion occurs via increased expression of luminal BK channels.

Blunted IgE-mediated activation of mast cells in mice lacking the Ca2+-activated K+ channel KCa3.1.

Mast cell stimulation by Ag is followed by the opening of Ca(2+)-activated K(+) channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca(2+)-activated K(+) channel K(Ca)3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of K(Ca)3.1 knockout mice (K(Ca)3.1(-/-)) and their wild-type littermates (K(Ca)3.1(+/+)). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcepsilonRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in K(Ca)3.1(-/-) BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca(2+) ionophore ionomycin was followed by stimulation of Ca(2+)-activated K(+) channels and cell membrane hyperpolarization in K(Ca)3.1(+/+), but not in K(Ca)3.1(-/-) BMMCs. Upon Ag stimulation, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in K(Ca)3.1(-/-) BMMCs. Similarly, Ca(2+) entry upon endothelin-1 stimulation was significantly reduced in K(Ca)3.1(-/-) cells. Ag-induced release of beta-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in K(Ca)3.1(-/-) BMMCs compared with K(Ca)3.1(+/+) BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in K(Ca)3.1(-/-) cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by approximately 50%) blunted in K(Ca)3.1(-/-) mice. In conclusion, K(Ca)3.1 is required for Ca(2+)-activated K(+) channel activity and Ca(2+)-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.

M2 muscarinic receptors induce airway smooth muscle activation via a dual, Gbetagamma-mediated inhibition of large conductance Ca2+-activated K+ channel activity.

Airway smooth muscle is richly endowed with muscarinic receptors of the M(2) and M(3) subtype. Stimulation of these receptors inhibits large conductance calcium-activated K(+) (BK) channels, a negative feed back regulator, in a pertussis toxin-sensitive manner and thus facilitates contraction. The underlying mechanism, however, is unknown. We therefore studied the activity of bovine trachea BK channels in HEK293 cells expressing the M(2) or M(3) receptor (M(2)R or M(3)R). In M(2)R- but not M(3)R-expressing cells, maximal effective concentrations of carbamoylcholine (CCh) inhibited whole cell BK currents by 53%. This M(2)R-induced inhibition was abolished by pertussis toxin treatment or overexpression of the Gbetagamma scavenger transducin-alpha. In inside-out patches, direct application of 300 nm purified Gbetagamma decreased channel open probability by 55%. The physical interaction of Gbetagamma with BK channels was confirmed by co-immunoprecipitation. Interestingly, inhibition of phospholipase C as well as protein kinase C activities also reversed the CCh effect but to a smaller (approximately 20%) extent. Mouse tracheal cells responded similarly to CCh, purified Gbetagamma and phospholipase C/protein kinase C inhibition as M(2)R-expressing HEK293 cells. Our results demonstrate that airway M(2)Rs inhibit BK channels by a dual, Gbetagamma-mediated mechanism, a direct membrane-delimited interaction, and the activation of the phospholipase C/protein kinase C pathway.

Cysteine-rich protein 2, a novel downstream effector of cGMP/cGMP-dependent protein kinase I-mediated persistent inflammatory pain.

The cGMP/cGMP-dependent protein kinase I (cGKI) signaling pathway plays an important role in spinal nociceptive processing. However, downstream targets of cGKI in this context have not been identified to date. Using a yeast two-hybrid screen, we isolated cysteine-rich protein 2 (CRP2) as a novel cGKI interactor in the spinal cord. CRP2 is expressed in laminas I and II of the mouse spinal cord and is colocalized with cGKI, calcitonin gene-related peptide, and isolectin B4. Moreover, the majority of CRP2 mRNA-positive dorsal root ganglion (DRG) neurons express cGKI and peripherin. CRP2 is phosphorylated in a cGMP-dependent manner, and its expression increases in the spinal cord and in DRGs after noxious stimulation of a hindpaw. To elucidate the functional role of CRP2 in nociception, we analyzed mice with a targeted deletion of CRP2. CRP2-deficient (CRP2-/-) mice demonstrate normal behavioral responses to acute nociception and after axonal injury of the sciatic nerve, but increased nociceptive behavior in models of inflammatory hyperalgesia compared with wild-type mice. Intrathecal administration of cGMP analogs increases the nociceptive behavior in wild-type but not in CRP2-/- mice, indicating that the presence of CRP2 is important for cGMP-mediated nociception. These data suggest that CRP2 is a new downstream effector of cGKI-mediated spinal nociceptive processing and point to an inhibitory role of CRP2 in the generation of inflammatory pain.

Hypothalamic-pituitary-adrenal axis hyporesponsiveness to restraint stress in mice deficient for large-conductance calcium- and voltage-activated potassium (BK) channels.

Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, releasing ACTH from the anterior pituitary gland and glucocorticoids from the adrenal cortex. Stress also activates the sympathetic nervous system, evoking adrenaline release from the adrenal medulla. Large-conductance calcium- and voltage-activated potassium (BK) channels have been implicated in regulation of cellular excitability in these systems. Here, we examine the functional role of BK channels in HPA axis regulation in vivo using female mice genetically deficient (BK(-/-)) for the pore-forming subunits of BK channels. BK(-/-) phenotype in the HPA was confirmed by immunohistochemistry, Western blot analysis, and corticotrope patch-clamp recording. Restraint stress-induced plasma concentrations of ACTH and corticosterone were significantly blunted in BK(-/-) mice compared with wild type (WT) controls. This stress hyporesponsiveness was associated with reduced activation of hypothalamic paraventricular nucleus (PVN) neurons. Basal expression of CRH, but not arginine vasopressin mRNA in the PVN was significantly lower in BK(-/-) mice compared with WT controls. Total anterior pituitary ACTH peptide content, but not proopiomelanocortin mRNA expression or corticotrope number, was significantly reduced in BK(-/-) mice compared with WT. However, anterior pituitary corticotropes from BK(-/-) mice fully supported ACTH output, releasing a significantly greater proportion of stored ACTH in response to secretagogue in vitro compared with WT. These results support an important role for BK channels in both the neural circuitry and endocrine output of the HPA axis and indicate that the stress hyporesponsiveness in BK(-/-) mice primarily results from reduced activation of hypothalamic PVN neurosecretory neurons.

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

The unique voltage- and Ca2+-dependent 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 carbachol concentration-dependently increased the tonic tension of wild-type (WT) trachea, mutant trachea showed a different response with rapid tension development followed by phasic contractions superimposed on a tonic component. Tonic contractions were substantially 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 microM. 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-depth 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.

Immunolocalization of BK channels in hippocampal pyramidal neurons.

Neurons are highly specialized cells in which the integration and processing of electrical signals critically depends on the precise localization of ion channels. For large-conductance Ca(2+)- activated K(+) (BK) channels, targeting to presynaptic membranes in hippocampal pyramidal cells was reported; however, functional evidence also suggests a somatodendritic localization. Therefore we re-examined the subcellular distribution of BK channels in mouse hippocampus using a panel of independent antibodies in a combined approach of conventional immunocytochemistry on cultured neurons, pre- and postembedding electron microscopy and immunoprecipitation. In cultured murine hippocampal neurons, the colocalization of BK channels with both pre- and postsynaptic marker proteins was observed. Electron microscopy confirmed targeting of BK channels to axonal as well as dendritic membranes of glutamatergic synapses in hippocampus. A postsynaptic localization of BK channels was also supported by the finding that the channel coimmunoprecipitated with PSD95, a protein solely expressed in the postsynaptic compartment. These results thus demonstrate that BK channels reside in both post- and presynaptic compartments of hippocampal pyramidal neurons.

Distal colonic K(+) secretion occurs via BK channels.

K(+) secretion in the kidney and distal colon is a main determinant of K(+) homeostasis. This study investigated the identity of the relevant luminal secretory K(+) ion channel in distal colon. An Ussing chamber was used to measure ion transport in the recently generated BK channel-deficient (BK(-/-)) mice. BK(-/-) mice display a significant colonic epithelial phenotype with (1) lack of Ba(2+)-sensitive resting K(+) secretion, (2) absence of K(+) secretion stimulated by luminal P2Y(2) and P2Y(4) receptors, (3) absence of luminal Ca(2+) ionophore (A23187)-stimulated K(+) secretion, (4) reduced K(+) and increased Na(+) contents in feces, and (5) an increased colonic Na(+) absorption. In contrast, resting and uridine triphosphate (UTP)-stimulated K(+) secretion was not altered in mice that were deficient for the intermediate conductance Ca(2+)-activated K(+) channel SK4. BK channels localize to the luminal membrane of crypt, and reverse transcription-PCR results confirm the expression of the BK channel alpha-subunit in isolated distal colonic crypts. It is concluded that BK channels are the responsible K(+) channels for resting and stimulated Ca(2+)-activated K(+) secretion in mouse distal colon.

Ca2+ -activated K+ channels of the BK-type in the mouse brain.

An antibody against the 442 carboxy-terminal amino acids of the BK channel alpha-subunit detects high immunoreactivity within the telencephalon in cerebral cortices, olfactory bulb, basal ganglia and hippocampus, while lower levels are found in basal forebrain regions and amygdala. Within the diencephalon, high density was found in nuclei of the ventral and dorsal thalamus and the medial habenular nucleus, and low density in the hypothalamus. The fasciculus retroflexus and its termination in the mesencephalic interpeduncular nucleus are prominently stained. Other mesencephalic expression sites are periaquaeductal gray and raphe nuclei. In the rhombencephalon, BK channels are enriched in the cerebellar cortex and in the locus coeruleus. Strong immunoreactivity is also contained in the vestibular nuclei, but not in cranial nerves and their intramedullary course of their roots. On the cellular level, BK channels show pre- and postsynaptic localizations, i.e., in somata, dendrites, axons and synaptic terminals.

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 alpha 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.

Melatonin receptor signaling in pregnant and nonpregnant rat uterine myocytes as probed by large conductance Ca2+-activated K+ channel activity.

The mRNAs of MT1 and MT2 melatonin receptors are present in cells from nonpregnant (NPM) and pregnant (PM) rat myometrium. To investigate the coupling of melatonin receptors to Gq- and Gi-type of heterotrimeric G proteins, we analyzed the activity of large-conductance Ca2+-activated K+ (BKCa) channels, the expression of which in the uterus is confined to smooth muscle cells. The melatonin receptor agonist 2-iodomelatonin induced a pertussis toxin (PTX)-insensitive increase in channel open probability that was blocked by the nonselective antagonist luzindole. The 2-iodomelatonin effect on channel open probability was suppressed by overexpression of the Gqalpha-inactivating protein RGS16 and the phospholipase C inhibitor U-73122. The activity of BKCa channels is differentially regulated by protein kinase A (PKA) in NPM and PM cells. Thus, the beta-adrenoceptor agonist isoprenaline inhibited the BKCa channel conducted whole-cell outward current (Iout) in NPM cells and enhanced Iout in PM cells. Additional application of 2-iodomelatonin antagonized the isoprenaline effect on Iout in NPM cells but enhanced Iout in PM cells. All 2-iodomelatonin effects on Iout were sensitive to PTX treatment and the PKA inhibitor H-89. We therefore conclude that melatonin activates both the PTX-insensitive Gq/phospholipase C/Ca2+ and the PTX-sensitive Gi/cAMP/PKA signaling pathway in rat myometrium.