Calcium-activated potassium channel family in coronary artery bypass grafts.

OBJECTIVES: We examined the expression, distribution, and contribution to vasodilatation of the calcium-activated potassium (KCa) channel family in the commonly used coronary artery bypass graft internal thoracic artery (ITA) and saphenous vein (SV) to understand the role of large conductance KCa (BKCa), intermediate-conductance KCa (IKCa), and small-conductance KCa (SKCa) channel subtypes in graft dilating properties determined by endothelium-smooth muscle interaction that is essential to the postoperative performance of the graft. METHODS: Real-time polymerase chain reaction and western blot were employed to detect the messenger RNA and protein level of KCa channel subtypes. Distribution of KCa channel subtypes was examined by immunohistochemistry. KCa subtype-mediated vasorelaxation was studied using wire myography. RESULTS: Both ITA and SV express all KCa channel subtypes with each subtype distributed in both endothelium and smooth muscle. ITA and SV do not differ in the overall expression level of each KCa channel subtype, corresponding to comparable relaxant responses to respective subtype activators. In ITA, BKCa is more abundantly expressed in smooth muscle than in endothelium, whereas SKCa exhibits more abundance in the endothelium. In comparison, SV shows even distribution of KCa channel subtypes in the 2 layers. The BKCa subtype in the KCa family plays a significant role in vasodilatation of ITA, whereas its contribution in SV is quite limited. CONCLUSIONS: KCa family is abundantly expressed in ITA and SV. There are differences between these 2 grafts in the abundance of KCa channel subtypes in the endothelium and the smooth muscle. The significance of the BKCa subtype in vasodilatation of ITA may suggest the potential of development of BKCa modulators for the prevention and treatment of ITA spasm during/after coronary artery bypass graft surgery.

Can endothelial hemoglobin-α regulate nitric oxide vasodilatory signaling?

We used mathematical modeling to investigate nitric oxide (NO)-dependent vasodilatory signaling in the arteriolar wall. Detailed continuum cellular models of calcium (Ca2+) dynamics and membrane electrophysiology in smooth muscle and endothelial cells (EC) were coupled with models of NO signaling and biotransport in an arteriole. We used this theoretical approach to examine the role of endothelial hemoglobin-α (Hbα) as a modulator of NO-mediated myoendothelial feedback, as previously suggested in Straub et al. (Nature 491: 473-477, 2012). The model considers enriched expression of inositol 1,4,5-triphosphate receptors (IP3Rs), endothelial nitric oxide synthase (eNOS) enzyme, Ca2+-activated potassium (KCa) channels and Hbα in myoendothelial projections (MPs) between the two cell layers. The model suggests that NO-mediated myoendothelial feedback is plausible if a significant percentage of eNOS is localized within or near the myoendothelial projection. Model results show that the ability of Hbα to regulate the myoendothelial feedback is conditional to its colocalization with eNOS near MPs at concentrations in the high nanomolar range (>0.2 µM or 24,000 molecules). Simulations also show that the effect of Hbα observed in in vitro experimental studies may overestimate its contribution in vivo, in the presence of blood perfusion. Thus, additional experimentation is required to quantify the presence and spatial distribution of Hbα in the EC, as well as to test that the strong effect of Hbα on NO signaling seen in vitro, translates also into a physiologically relevant response in vivo.NEW & NOTEWORTHY Mathematical modeling shows that although regulation of nitric oxide signaling by hemoglobin-α (Hbα) is plausible, it is conditional to its presence in significant concentrations colocalized with endothelial nitric oxide synthase in myoendothelial projections. Additional experimentation is required to test that the strong effect of Hbα seen in vitro translates into a physiologically relevant response in vivo.

Upregulation of the large conductance voltage- and Ca2+-activated K+ channels by Janus kinase 2.

The iberiotoxin-sensitive large conductance voltage- and Ca(2+)-activated potassium (BK) channels (maxi-K(+)-channels) hyperpolarize the cell membrane thus supporting Ca(2+) entry through Ca(2+)-release activated Ca(2+) channels. Janus kinase-2 (JAK2) has been identified as novel regulator of ion transport. To explore whether JAK2 participates in the regulation of BK channels, cRNA encoding Ca(2+)-insensitive BK channels (BK(M513I+Δ899-903)) was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, gain-of-function (V617F)JAK2, or inactive (K882E)JAK2. K(+) conductance was determined by dual electrode voltage clamp and BK-channel protein abundance by confocal microscopy. In A204 alveolar rhabdomyosarcoma cells, iberiotoxin-sensitive K(+) current was determined utilizing whole cell patch clamp. A204 cells were further transfected with JAK2 and BK-channel transcript, and protein abundance was quantified by RT-PCR and Western blotting, respectively. As a result, the K(+) current in BK(M513I+Δ899-903)-expressing oocytes was significantly increased following coexpression of JAK2 or (V617F)JAK2 but not (K882E)JAK2. Coexpression of the BK channel with (V617F)JAK2 but not (K882E)JAK2 enhanced BK-channel protein abundance in the oocyte cell membrane. Exposure of BK-channel and (V617F)JAK2-expressing oocytes to the JAK2 inhibitor AG490 (40 µM) significantly decreased K(+) current. Inhibition of channel insertion by brefeldin A (5 µM) decreased the K(+) current to a similar extent in oocytes expressing the BK channel alone and in oocytes expressing the BK channel and (V617F)JAK2. The iberiotoxin (50 nM)-sensitive K(+) current in rhabdomyosarcoma cells was significantly decreased by AG490 pretreatment (40 µM, 12 h). Moreover, overexpression of JAK2 in A204 cells significantly enhanced BK channel mRNA and protein abundance. In conclusion, JAK2 upregulates BK channels by increasing channel protein abundance in the cell membrane.

Calcium activated potassium channel expression during human iPS cell-derived neurogenesis.

The family of calcium activated potassium channels of low and intermediate conductance, known as SK channels, consists of four members (SK1-4). These channels are widely expressed throughout the organism and involved in various cellular processes, such as the afterhyperpolarization in excitable cells but also in differentiation processes of various tissues. To date, the role of SK channels in developmental processes has been merely a marginal focus of investigation, although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently, several studies from our laboratory delineated the influence of SK channel expression and their respective activity on cytoskeletal reorganization in neural and pluripotent stem cells and regulation of cell fate determination toward the cardiac lineage in human and mouse pluripotent stem cells. Herein, we have now analyzed SK channel expression patterns and distribution at various stages of human induced pluripotent stem cell-derived neurogenesis particularly focusing on undifferentiated iPS cells, neural progenitors and mature neurons. All family members could be detected starting at the iPS cell level and were differentially expressed during the subsequent maturation process. Intriguingly, we found obvious discrepancies between mRNA and protein expression pointing toward a complex regulatory mechanism. Inhibition of SK channels with either apamin or clotrimazol did not have any significant effects on the speed or amount of neurogenesis in vitro. The abundance and specific regulation of SK channel expression during iPS cell differentiation indicates distinct roles of these ion channels not only for the cardiac but also for neuronal cell differentiation and in vitro neurogenesis.

Voltage-dependent sodium channels and calcium-activated potassium channels in human odontoblasts in vitro.

INTRODUCTION: Transmembrane ionic signaling regulates many cellular processes in both physiological and pathologic settings. In this study, the biophysical properties of voltage-dependent Na(+) channels in odontoblasts derived from human dental pulp (HOB cells) were investigated together with the effect of bradykinin on intracellular Ca(2+) signaling and expression of Ca(2+)-activated K(+) channels. METHODS: Ionic channel activity was characterized by using whole-cell patch-clamp recording and fura-2 fluorescence. RESULTS: Mean resting membrane potential in the HOB cells was -38 mV. Depolarizing steps from a holding potential of -80 mV activated transient voltage-dependent inward currents with rapid activation/inactivation properties. At a holding potential of -50 mV, no inward current was recorded. Fast-activation kinetics exhibited dependence on membrane potential, whereas fast-inactivation kinetics did not. Steady-state inactivation was described by a Boltzmann function with a half-maximal inactivation potential of -70 mV, indicating that whereas the channels were completely inactivated at physiological resting membrane potential, they could be activated when the cells were hyperpolarized. Inward currents disappeared in Na(+)-free extracellular solution. Bradykinin activated intracellular Ca(2+)-releasing and influx pathways. When the HOB cells were clamped at a holding potential of -50 mV, outward currents were recorded at positive potentials, indicating sensitivity to inhibitors of intermediate-conductance Ca(2+)-activated K(+) channels. CONCLUSIONS: Human odontoblasts expressed voltage-dependent Na(+) channels, bradykinin receptors, and Ca(2+)-activated K(+) channels, which play an important role in driving cellular functions by channel-receptor signal interaction and membrane potential regulation.

Twenty-four-hour exposure to altered blood flow modifies endothelial Ca2+-activated K+ channels in rat mesenteric arteries.

We tested the hypothesis that changes in arterial blood flow modify the function of endothelial Ca2+-activated K+ channels [calcium-activated K+ channel (K(Ca)), small-conductance calcium-activated K+ channel (SK3), and intermediate calcium-activated K+ channel (IK1)] before arterial structural remodeling. In rats, mesenteric arteries were exposed to increased [+90%, high flow (HF)] or reduced blood flow [-90%, low flow (LF)] and analyzed 24 h later. There were no detectable changes in arterial structure or in expression level of endothelial nitric-oxide synthase, SK3, or IK1. Arterial relaxing responses to acetylcholine and 3-oxime-6,7-dichlore-1H-indole-2,3-dione (NS309; activator of SK3 and IK1) were measured in the absence and presence of endothelium, NO, and prostanoid blockers, and 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-metheno-7H-dibenzo [b,n] [1,5,12,16]tetraazacyclotricosine-5,13-diium dibromide (UCL 1684; inhibitor of SK3) or 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; inhibitor of IK1). In LF arteries, endothelium-dependent relaxation was markedly reduced, due to a reduction in the endothelium-derived hyperpolarizing factor (EDHF) response. In HF arteries, the balance between the NO/prostanoid versus EDHF response was unaltered. However, the contribution of IK1 to the EDHF response was enhanced, as indicated by a larger effect of TRAM-34 and a larger residual NS309-induced relaxation in the presence of UCL 1684. Reduction of blood flow selectively blunts EDHF relaxation in resistance arteries through inhibition of the function of K(Ca) channels. An increase in blood flow leads to a more prominent role of IK1 channels in this relaxation.

Impaired mast cell activation in gene-targeted mice lacking the serum- and glucocorticoid-inducible kinase SGK1.

The PI3K pathway plays a pivotal role in the stimulation of mast cells. PI3K-dependent kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored the role of SGK1 in mast cell function. Mast cells were isolated from bone marrow (BMMC) of SGK1 knockout mice (sgk1(-/-)) and their wild-type littermates (sgk1(+/+)). The BMMC number as well as CD117, CD34, and FcepsilonRI expression in BMCCs were similar in both genotypes. Upon Ag stimulation of the FcepsilonRI receptor, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in sgk1(-/-) BMMCs. The currents through Ca(2+)-activated K+ channels induced by Ag were significantly higher in sgk1(+/+) BMMCs than in sgk1(-/-) BMMCs. Treatment with the Ca(2+) ionophore ionomycin (1 microM) led to activation of the K+ channels in both genotypes, indicating that the Ca(2+)-activated K+ channels are similarly expressed and sensitive to activation by Ca(2+) in sgk1(+/+) and sgk1(-/-) BMMCs, and that blunted stimulation of Ca(2+)-activated K+ channels was secondary to decreased Ca(2+) entry. Ag-IgE-induced degranulation and early IL-6 secretion were also significantly blunted in sgk1(-/-) BMMCs. The decrease in body temperature following Ag treatment, which reflects an anaphylactic reaction, was substantially reduced in sgk1(-/-) mice, pointing to impaired mast cell function in vivo. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk1(-/-) than in sgk1(+/+)mice. The observations reveal a critical role for SGK1 in ion channel regulation and the function of mast cells, and thus disclose a completely novel player in the regulation of allergic reaction.

Endothelial Ca+-activated K+ channels in normal and impaired EDHF-dilator responses--relevance to cardiovascular pathologies and drug discovery.

The arterial endothelium critically contributes to blood pressure control by releasing vasodilating autacoids such as nitric oxide, prostacyclin and a third factor or pathway termed 'endothelium-derived hyperpolarizing factor' (EDHF). The nature of EDHF and EDHF-signalling pathways is not fully understood yet. However, endothelial hyperpolarization mediated by the Ca(2+)-activated K(+) channels (K(Ca)) has been suggested to play a critical role in initializing EDHF-dilator responses in conduit and resistance-sized arteries of many species including humans. Endothelial K(Ca) currents are mediated by the two K(Ca) subtypes, intermediate-conductance K(Ca) (KCa3.1) (also known as, a.k.a. IK(Ca)) and small-conductance K(Ca) type 3 (KCa2.3) (a.k.a. SK(Ca)). In this review, we summarize current knowledge about endothelial KCa3.1 and KCa2.3 channels, their molecular and pharmacological properties and their specific roles in endothelial function and, particularly, in the EDHF-dilator response. In addition we focus on recent experimental evidences derived from KCa3.1- and/or KCa2.3-deficient mice that exhibit severe defects in EDHF signalling and elevated blood pressures, thus highlighting the importance of the KCa3.1/KCa2.3-EDHF-dilator system for blood pressure control. Moreover, we outline differential and overlapping roles of KCa3.1 and KCa2.3 for EDHF signalling as well as for nitric oxide synthesis and discuss recent evidence for a heterogeneous (sub) cellular distribution of KCa3.1 (at endothelial projections towards the smooth muscle) and KCa2.3 (at inter-endothelial borders and caveolae), which may explain their distinct roles for endothelial function. Finally, we summarize the interrelations of altered KCa3.1/KCa2.3 and EDHF system impairments with cardiovascular disease states such as hypertension, diabetes, dyslipidemia and atherosclerosis and discuss the therapeutic potential of KCa3.1/KCa2.3 openers as novel types of blood pressure-lowering drugs.

CREB regulation of BK channel gene expression underlies rapid drug tolerance.

Pharmacodynamic tolerance is believed to involve homeostatic mechanisms initiated to restore normal neural function. Drosophila exposed to a sedating dose of an organic solvent, such as benzyl alcohol or ethanol, acquire tolerance to subsequent sedation by that solvent. The slo gene encodes BK-type Ca(2+)-activated K(+) channels and has been linked to alcohol- and organic solvent-induced behavioral tolerance in mice, Caenorhabditis elegans (C. elegans) and Drosophila. The cyclic AMP response element-binding (CREB) proteins are transcription factors that have been mechanistically linked to some behavioral changes associated with drug addiction. Here, we show that benzyl alcohol sedation alters expression of both dCREB-A and dCREB2-b genes to increase production of positively acting CREB isoforms and to reduce expression of negatively acting CREB variants. Using a CREB-responsive reporter gene, we show that benzyl alcohol sedation increases CREB-mediated transcription. Chromatin immunoprecipitation assays show that the binding of dCREB2, with a phosphorylated kinase-inducible domain, increases immediately after benzyl alcohol sedation within the slo promoter region. Most importantly, we show that a loss-of-function allele of dCREB2 eliminates drug-induced upregulation of slo expression and the production of benzyl alcohol tolerance. This unambiguously links dCREB2 transcription factors to these two benzyl alcohol-induced phenotypes. These findings suggest that CREB positively regulates the expression of slo-encoded BK-type Ca(2+)-activated K(+) channels and that this gives rise to behavioral tolerance to benzyl alcohol sedation.

A novel actin-binding domain on Slo1 calcium-activated potassium channels is necessary for their expression in the plasma membrane.

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels regulate the physiological properties of many cell types. The gating properties of BK(Ca) channels are Ca(2+)-, voltage- and stretch-sensitive, and stretch-sensitive gating of these channels requires interactions with actin microfilaments subjacent to the plasma membrane. Moreover, we have previously shown that trafficking of BK(Ca) channels to the plasma membrane is associated with processes that alter cytoskeletal dynamics. Here, we show that the Slo1 subunits of BK(Ca) channels contain a novel cytoplasmic actin-binding domain (ABD) close to the C terminus, considerably downstream from regions of the channel molecule that play a major role in determining channel-gating properties. Binding of actin to the ABD can occur in a binary mixture in the absence of other proteins. Coexpression of a small ABD-green fluorescent protein fusion protein that competes with full-length Slo1 channels for binding to actin markedly suppresses trafficking of full-length Slo1 channels to the plasma membrane. In addition, Slo1 channels containing deletions of the ABD that eliminate actin binding are retained in intracellular pools, and they are not expressed on the cell surface. At least one point mutation within the ABD (L1020A) reduces surface expression of Slo1 channels to approximately 25% of wild type, but it does not cause a marked effect on the gating of point mutant channels that reach the cell surface. These data suggest that Slo1-actin interactions are necessary for normal trafficking of BK(Ca) channels to the plasma membrane and that the mechanisms of this interaction may be different from those that underlie F-actin and stretch-sensitive gating.

The effect of neurotrophic factors on morphology, TRPV1 expression and capsaicin responses of cultured human DRG sensory neurons.

We have studied the effect of key neurotrophic factors (NTFs) on morphology, levels of the vanilloid receptor-1 (TRPV1) and responses to capsaicin in adult human sensory neurons in vitro. Avulsed dorsal root ganglia (DRG, n = 5) were cultured with or without a combination of nerve growth factor (NGF), glial cell (line)-derived growth factor (GDNF) and neurotrophin3 (NT3) for 5 days. In the absence of NTFs, the diameter of neurons ranged from 20 to 100 microm (mean 42 +/- 4 microm). Adding NTFs caused a significant increase in neuronal sizes, up to 120 microm (mean diameter 62 +/- 5 microm, P < 0.01, t-test), an overall 35% increase of TRPV1-positive neurons (P < 0.003), and notably of large TRPV1-positive neurons > 80 microm (P < 0.05). Responses to capsaicin were significantly enhanced with calcium ratiometry (P < 0.0001). Short duration (1h) exposure of dissociated sensory neurons to NTFs increased numbers of TRPV1-positive neurons, but not of TRPV3, Nav 1.8 and IK1 and the morphological size-distribution remained similar to intact post-mortem DRG neurons. NTFs thus increase size, elevate TRPV1 levels and enhance capsaicin responses in cultured human DRG neurons; these changes may relate to pathophysiology in disease states, and provide an in vitro model to study novel analgesics.

Role of the C-terminus of the high-conductance calcium-activated potassium channel in channel structure and function.

The role of ion channels in cell physiology is regulated by processes occurring after protein biosynthesis, which are critical for both channel function and targeting of channels to appropriate cell compartments. Here we apply biochemical and electrophysiological methods to investigate the role of the high-conductance, calcium-activated potassium (Maxi-K) channel C-terminal domain in channel tetramerization, association with the beta1 subunit, trafficking of the channel complex to the cell surface, and channel function. No evidence for channel tetramerization, cell surface expression, or function was observed with Maxi-K(1)(-)(323), a construct truncated three residues after the S(6) transmembrane domain. However, Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) are able to form tetramers and to associate with the beta1 subunit. Maxi-K(1)(-)(343)-beta1 and Maxi-K(1)(-)(441)-beta1 complexes are efficiently targeted to the cell surface and cannot be pharmacologically distinguished from full-length channels in binding experiments but do not form functional channels. Maxi-K(1)(-)(651) forms tetramers and associates with beta1; however, the complex is not present at the cell surface, but is retained intracellularly. Maxi-K(1)(-)(651) surface expression and channel function can be fully rescued after coexpression with its C-terminal complement, Maxi-K(652)(-)(1113). However coexpression of Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) with their respective C-terminal complements did not rescue channel function. Together, these data demonstrate that the domain(s) in the Maxi-K channel necessary for formation of tetramers, coassembly with the beta1 subunit, and cell surface expression resides within the S(0)-S(6) linker domain of the protein, and that structural constraints within the gating ring in the C-terminal region can regulate trafficking and function of constructs truncated in this region.

Dietary K+ regulates apical membrane expression of maxi-K channels in rabbit cortical collecting duct.

The cortical collecting duct (CCD) is a final site for regulation of K(+) homeostasis. CCD K(+) secretion is determined by the electrochemical gradient and apical permeability to K(+). Conducting secretory K(+) (SK/ROMK) and maxi-K channels are present in the apical membrane of the CCD, the former in principal cells and the latter in both principal and intercalated cells. Whereas SK channels mediate baseline K(+) secretion, maxi-K channels appear to participate in flow-stimulated K(+) secretion. Chronic dietary K(+) loading enhances the CCD K(+) secretory capacity due, in part, to an increase in SK channel density (Palmer et al., J Gen Physiol 104: 693-710, 1994). Long-term exposure of Ambystoma tigrinum to elevated K(+) increases renal K(+) excretion due to an increase in apical maxi-K channel density in their CDs (Stoner and Viggiano, J Membr Biol 162: 107-116, 1998). The purpose of the present study was to test whether K(+) adaptation in the mammalian CCD is associated with upregulation of maxi-K channel expression. New Zealand White rabbits were fed a low (LK), control (CK), or high (HK) K(+) diet for 10-14 days. Real-time PCR quantitation of message encoding maxi-K alpha- and beta(2-4)-subunits in single CCDs from HK animals was greater than that detected in CK and LK animals (P < 0.05); beta(1)-subunit was not detected in any CCD sample but was present in whole kidney. Indirect immunofluorescence microscopy revealed a predominantly intracellular distribution of alpha-subunits in LK kidneys. In contrast, robust apical labeling was detected primarily in alpha-intercalated cells in HK kidneys. In summary, K(+) adaptation is associated with an increase in steady-state abundance of maxi-K channel subunit-specific mRNAs and immunodetectable apical alpha-subunit, the latter observation consistent with redistribution from an intracellular pool to the plasma membrane.

Accessory Kvbeta1 subunits differentially modulate the functional expression of voltage-gated K+ channels in mouse ventricular myocytes.

Voltage-gated K+ (Kv) channel accessory (beta) subunits associate with pore-forming Kv alpha subunits and modify the properties and/or cell surface expression of Kv channels in heterologous expression systems. There is very little presently known, however, about the functional role(s) of Kv beta subunits in the generation of native cardiac Kv channels. Exploiting mice with a targeted disruption of the Kvbeta1 gene (Kvbeta1-/-), the studies here were undertaken to explore directly the role of Kvbeta1 in the generation of ventricular Kv currents. Action potential waveforms and peak Kv current densities are indistinguishable in myocytes isolated from the left ventricular apex (LVA) of Kvbeta1-/- and wild-type (WT) animals. Analysis of Kv current waveforms, however, revealed that mean+/-SEM I(to,f) density is significantly (P< or =0.01) lower in Kvbeta1-/- (21.0+/-0.9 pA/pF; n=68), than in WT (25.3+/-1.4 pA/pF; n=42), LVA myocytes, and that mean+/-SEM I(K,slow) density is significantly (P< or =0.01) higher in Kvbeta1-/- (19.1+/-0.9 pA/pF; n=68), compared with WT (15.9+/-0.7 pA/pF; n=42), LVA cells. Pharmacological studies demonstrated that the TEA-sensitive component of I(K,slow), I(K,slow2,) is selectively increased in Kvbeta1-/- LVA myocytes. In parallel with the alterations in I(to,f) and I(K,slow2) densities, Kv4.3 expression is decreased and Kv2.1 expression is increased in Kvbeta1-/- ventricles. Taken together, these results demonstrate that Kvbeta1 differentially regulates the functional cell surface expression of myocardial I(to,f) and I(K,slow2) channels.

Effect of chronic cigarette smoking on large-conductance calcium-activated potassium channel and Kv1.5 expression in bronchial smooth muscle cells of rats.

To investigate the role of potassium channels in the pathogenesis of airway hyperresponsiveness induced by cigarette smoking, the alteration in expression of large-conductance calcium-activated potassium channel (BKca) and voltage-dependent delayed rectifier potassium channel (Kv1.5) in bronchial smooth muscle cells were investigated in chronic cigarette smoking rats. Airway responsiveness was determined, hematoxylin and eosin staining, immuno-histochemistry, in-situ hybridization and western blot techniques were used. The results showed: (1) Chronic cigarette smoking down-regulated the protein synthesis and mRNA expression of BKca and Kv1.5 in bronchial and bronchiolar smooth muscles. (2) BKca decreased more markedly than Kv1.5 in bronchi, but there was no difference between them in bronchioli. (3) No changes in the expression of these two potassium channel proteins were found in extracted cell membrane protein from lung tissue. The results suggest that chronic cigarette smoking can down-regulate the levels of BKca and Kv1.5 in rat bronchial smooth muscle cells in vivo, which might contribute to the mechanism of airway hyperresponsiveness induced by cigarette smoking.

Role for calcium-activated potassium channels (BK) in growth control of human malignant glioma cells.

Voltage-dependent large-conductance Ca(2+)-activated K(+) channels, often referred to as BK channels, are a unique class of ion channels coupling intracellular chemical signaling to electrical signaling. BK channel expression has been shown to be up-regulated in human glioma biopsies, and expression levels correlate positively with the malignancy grade of the tumor. Glioma BK channels (gBK) are a splice variant of the hslo gene, are characterized by enhanced sensitivity to [Ca(2+)](i), and are the target of modulation by growth factors. By using the selective pharmacological BK channel inhibitor iberiotoxin, we examined the potential role of these channels in tumor growth. Cell survival assays examined the ability of glioma cells to grow in nominally serum-free medium. Under such conditions, BK channel inhibition by iberiotoxin caused a dose- and time-dependent decrease in cell number discernible as early as 72 hr after exposure and maximal growth inhibition after 4-5 days. FACS analysis shows that IbTX treatment arrests glioma cells in S phase of the cell cycle, whereupon cells undergo cell death. Interestingly, IbTX effects were nullified when cells were maintained in 7% fetal calf serum. Electrophysiological analysis, in conjunction with biotinylation studies, demonstrates that serum starvation caused a significant translocation of BK channel protein to the plasma membrane, corresponding to a two- to threefold increase in whole-cell conductance, but without a change in total gBK protein. Hence, expression of functional gBK channels appears to be regulated in a growth-factor-dependent manner, with enhanced surface expression promoting tumor cell growth under conditions of growth factor deprivation as might occur under in vivo conditions.

Comparative immunohistochemical distribution of three small-conductance Ca2+-activated potassium channel subunits, SK1, SK2, and SK3 in mouse brain.

To investigate the distribution of all three SK channel subunits in the mouse central nervous system, we performed immunohistochemistry using sequence-specific antibodies directed against SK1, SK2, and SK3 proteins. Expression of SK1 and SK2 proteins revealed a partly overlapping distribution pattern restricted to a limited number of brain areas (e.g., neocortex, hippocampal formation). In contrast, SK3 immunoreactivity was rather complementary and predominantly detected in phylogenetically older brain regions like basal ganglia, thalamus, and various brain stem nuclei (e.g., locus coeruleus, tegmental nuclei). At the cellular level, SK1- and SK2-like immunoreactivity was primarily localized to somatic and dendritic structures, whereas the majority of SK3-like immunoreactivity was associated with varicose fibers.

Regional distribution of SK3 mRNA-containing neurons in the adult and adolescent rat ventral midbrain and their relationship to dopamine-containing cells.

SK3 small conductance, calcium-activated potassium channels play an important role in regulating the activity of mesencephalic dopamine (DA) neurons. In the present series of experiments, in situ hybridization techniques were used to compare SK3 and tyrosine hydroxylase (TH) mRNA expression throughout the rostrocaudal extent of the ventral midbrain in juvenile and adult rats. SK3 mRNA was found exclusively in areas that also contained large numbers of DA neurons including the substantia nigra (SN), the ventral tegmental area, and related cell groups (VTA-A10). An anteroposterior and mediolateral gradient in SK3 mRNA hybridization was apparent in the VTA-A10 but not in the SN. Younger rats appeared to possess higher levels and less regional variation in TH and SK3 transcripts. These results are consistent with previous studies reporting differential expression of SK3 protein within the midbrain and suggest that variations in SK3 channel distribution could contribute to differences in dopamine-related functions in the rat.

Small and intermediate conductance Ca(2+)-activated K+ channels confer distinctive patterns of distribution in human tissues and differential cellular localisation in the colon and corpus cavernosum.

The SK/IK family of small and intermediate conductance calcium-activated potassium channels contains four members, SK1, SK2, SK3 and IK1, and is important for the regulation of a variety of neuronal and non-neuronal functions. In this study we have analysed the distribution of these channels in human tissues and their cellular localisation in samples of colon and corpus cavernosum. SK1 mRNA was detected almost exclusively in neuronal tissues. SK2 mRNA distribution was restricted but more widespread than SK1, and was detected in adrenal gland, brain, prostate, bladder, liver and heart. SK3 mRNA was detected in almost every tissue examined. It was highly expressed in brain and in smooth muscle-rich tissues including the clitoris and the corpus cavernosum, and expression in the corpus cavernosum was upregulated up to 5-fold in patients undergoing sex-change operations. IK1 mRNA was present in surface-rich, secretory and inflammatory cell-rich tissues, highest in the trachea, prostate, placenta and salivary glands. In detailed immunohistochemical studies of the colon and the corpus cavernosum, SK1-like immunoreactivity was observed in the enteric neurons. SK3-like immunoreactivity was observed strongly in smooth muscle and vascular endothelium. IK1-like immunoreactivity was mainly observed in inflammatory cells and enteric neurons of the colon, but absent in corpus cavernosum. These distinctive patterns of distribution suggest that these channels are likely to have different biological functions and could be specifically targeted for a number of human diseases, such as irritable bowel syndrome, hypertension and erectile dysfunction.

Co-expression of mCysLT1 receptors and IK channels in Xenopus laevis oocytes elicits LTD4-stimulated IK current, independent of an increase in [Ca2+]i.

Addition of LTD4 (10 nM) to Xenopus laevis oocytes expressing the mCysLT1 receptor together with hBK or hIK channels resulted in the activation of both channels secondary to an LTD4-induced increase in [Ca2+]i. In addition, the hIK channel is activated by low concentrations of LTD4 (<0.1 nM), which did not result in any increase in [Ca2+]i. Even though activation of hIK by low concentrations of LTD4 was independent of an increase in [Ca2+]i, a certain "permissive" level of [Ca2+]i was required for its activation, since buffering of intracellular Ca2+ by EGTA completely abolished the response to LTD4. Neither hTBAK1 nor hTASK2 was activated following stimulations with LTD4 (0.1 and 100 nM).