Gamma-dendrotoxin blocks large conductance Ca2+-activated K+ channels in neuroblastoma cells

In N1E 115 neuroblastoma cells, gamma-dendrotoxin (DTX, 200 nM) blocked the outward K(+) current by 31.1 +/- 3.5% (n = 4) with approximately 500 nM Ca(2+) in the pipet solution, but had no effect on the outward K(+) current when internal Ca(2+) was reduced. Using a ramp protocol, iberiotoxin (IbTX, 100 nM) inhibited a component of the whole cell current, but in the presence of 200 nM gamma-DTX, no further inhibition by IbTX was observed. Two types of single channels were seen using outside-out patches when the pipette free Ca(2+) concentration was approximately 500 nM; a 63 pS and a 187 pS channel. The 63 pS channel was TEA-, IbTX- and gamma-DTX-insensitive, while the 187 pS channel was blocked by 1 mM TEA, 100 nM IbTX or 200 nM gamma-DTX. Both channels were activated by external application of ionomycin, when the pipet calcium concentration was reduced. gamma-DTX (200 nM) reduced the probability of openings of the 187 pS channel, with an IC(50) of 8.5 nM. In GH(3) cells gamma-DTX (200 nM) also blocked an IbTX-sensitive component of whole-cell K(+) currents. These results suggest that gamma-DTX blocks a large conductance Ca(2+) activated K(+) current in N1E 115 cells. This is the first indication that any of the dendrotoxins, which have classically been known to block voltage-gated (Kv) channels, can also block Ca(2+) activated K(+) channels.     

Membrane-delimited inhibition of maxi-K channel activity by the intermediate conductance Ca2+-activated K channel

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.     

Effect of phosphatidylserine on unitary conductance and Ba2+ block of the BK Ca2+-activated K+ channel: re-examination of the surface charge hypothesis

Incorporation of BK Ca2+-activated K+ channels into planar bilayers composed of negatively charged phospholipids such as phosphatidylserine (PS) or phosphatidylinositol (PI) results in a large enhancement of unitary conductance (gch) in comparison to BK channels in bilayers formed from the neutral zwitterionic lipid, phospatidylethanolamine (PE). Enhancement of gch by PS or PI is inversely dependent on KCl concentration, decreasing from 70% at 10 mM KCl to 8% at 1,000 mM KCl. This effect was explained previously by a surface charge hypothesis (Moczydlowski, E., O. Alvarez, C. Vergara, and R. Latorre. 1985. J. Membr. Biol. 83:273-282), which attributed the conductance enhancement to an increase in local K+ concentration near the entryways of the channel. To test this hypothesis, we measured the kinetics of block by external and internal Ba2+, a divalent cation that is expected to respond strongly to changes in surface electrostatics. We observed little or no effect of PS on discrete blocking kinetics by external and internal Ba2+ at 100 mM KCl and only a small enhancement of discrete and fast block by external Ba2+ in PS-containing membranes at 20 mM KCl. Model calculations of effective surface potential sensed by the K+ conduction and Ba2+-blocking reactions using the Gouy-Chapman-Stern theory of lipid surface charge do not lend support to a simple electrostatic mechanism that predicts valence-dependent increase of local cation concentration. The results imply that the conduction pore of the BK channel is electrostatically insulated from the lipid surface, presumably by a lateral distance of separation (>20 A) from the lipid head groups. The lack of effect of PS on apparent association and dissociation rates of Ba2+ suggest that lipid modulation of K+ conductance is preferentially coupled through conformational changes of the selectivity filter region that determine the high K+ flux rate of this channel relative to other cations. We discuss possible mechanisms for the effect of anionic lipids in the context of specific molecular interactions of phospholipids documented for the KcsA bacterial potassium channel and general membrane physical properties proposed to regulate membrane protein conformation via energetics of bilayer stress.     

Ca2+-dependent gating mechanisms for dSlo, a large-conductance Ca2+-activated K+ (BK) channel.

The Ca2+-dependent gating mechanism of cloned BK channels from Drosophila (dSlo) was studied. Both a natural variant (A1/C2/E1/G3/IO) and a mutant (S942A) were expressed in Xenopus oocytes, and single-channel currents were recorded from excised patches of membrane. Stability plots were used to define stable segments of data. Unlike native BK channels from rat skeletal muscle in which increasing internal Ca2+ concentration (Cai2+) in the range of 5 to 30 microM increases mean open time, increasing Cai2+ in this range for dSlo had little effect on mean open time. However, further increases in Cai2+ to 300 or 3000 microM then typically increased dSlo mean open time. Kinetic schemes for the observed Ca2+-dependent gating kinetics of dSlo were evaluated by fitting two-dimensional dwell-time distributions using maximum likelihood techniques and by comparing observed dependency plots with those predicted by the models. Previously described kinetic schemes that largely account for the Ca2+-dependent kinetics of native BK channels from rat skeletal muscle did not adequately describe the Ca2+ dependence of dSlo. An expanded version of these schemes which, in addition to the Ca2+-activation steps, permitted a Ca2+-facilitated transition from each open state to a closed state, could approximate the Ca2+-dependent kinetics of dSlo, suggesting that Ca2+ may exert dual effects on gating.     

Nitric oxide directly activates large conductance Ca2+-activated K+ channels (rSlo)

We investigated whether nitric oxide (NO) directly activates the cloned alpha-subunit of large conductance Ca2+-activated K+ (Maxi-K) channels from rat brain (rSlo), expressed either in HEK293 cells or Xenopus oocytes. In inside-out patches, the application of S-nitroso-N-acetylpenicillamine (SNAP), a NO-releasing compound, reversibly activated the channel shifting the voltage dependent activation curve of the macroscopic Maxi-K current to the left by about 15 mV. Pretreatment of the patches with N-ethylmaleimide to alkylate free sulfhydryl groups did not prevent the effect of SNAP, suggesting that NO may directly interact with the channels. These results suggest that Maxi-K channels might be one of the physiological targets of NO in the brain.     

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.     

Hydrophobic interface between two regulators of K+ conductance domains critical for calcium-dependent activation of large conductance Ca2+-activated K+ channels

It has been suggested that the large conductance Ca(2)+-activated K(+) channel contains one or more domains known as regulators of K(+) conductance (RCK) in its cytosolic C terminus. Here, we show that the second RCK domain (RCK2) is functionally important and that it forms a heterodimer with RCK1 via a hydrophobic interface. Mutant channels lacking RCK2 are nonfunctional despite their tetramerization and surface expression. The hydrophobic residues that are expected to form an interface between RCK1 and RCK2, based on the crystal structure of the bacterial MthK channel, are well conserved, and the interactions of these residues were confirmed by mutant cycle analysis. The hydrophobic interaction appears to be critical for the Ca(2+)-dependent gating of the large conductance Ca(2+)-activated K(+) channel.     

Expression and modulation of the intermediate- conductance Ca2+-activated K+ channel in glioblastoma GL-15 cells.

We report here the expression and properties of the intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) channel in the GL-15 human glioblastoma cell line. Macroscopic IK(Ca) currents on GL-15 cells displayed a mean amplitude of 7.2+/-0.8 pA/pF at 0 mV, at day 1 after plating. The current was inhibited by clotrimazole (CTL, IC(50)=257 nM), TRAM-34 (IC(50)=55 nM), and charybdotoxin (CTX, IC(50)=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IK(Ca) channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a K(D) for Ca(2+) of 188 nM at -100 mV, and no voltage dependence. We tested whether the IKCa channel expression in GL-15 cells could be the result of an increased ERK activity. Inhibition of the ERK pathway with the MEK antagonist PD98059 (25 muM, for 5 days) virtually suppressed the IK(Ca) current in GL-15 cells. PD98059 treatment also increased the length of cellular processes and up-regulated the astrocytic differentiative marker GFAP. A significant reduction of the IKCa current amplitude was also observed with time in culture, with mean currents of 7.17+/-0.75 pA/pF at 1-2 days, and 3.11+/-1.35 pA/pF at 5-6 days after plating. This time-dependent downregulation of the IK(Ca) current was not accompanied by changes in the ERK activity, as assessed by immunoblot analysis. Semiquantitative RT-PCR analysis demonstrated a ~35% reduction of the IK(Ca) channel mRNA resulting from ERK inhibition and a approximately 50% reduction with time in culture.     

Mode of action of iberiotoxin, a potent blocker of the large conductance Ca(2+)-activated K+ channel.

Iberiotoxin, a toxin purified from the scorpion Buthus tamulus is a 37 amino acid peptide having 68% homology with charybdotoxin. Charybdotoxin blocks large conductance Ca(2+)-activated K+ channels at nanomolar concentrations from the external side only (Miller, C., E. Moczydlowski, R. Latorre, and M. Phillips. 1985. Nature (Lond.). 313:316-318). Like charybdotoxin, iberiotoxin is only able to block the skeletal muscle membrane Ca(2+)-activated K+ channel incorporated into neutral-planar bilayers when applied to the external side. In the presence of iberiotoxin, channel activity is interrupted by quiescent periods that can last for several minutes. From single-channel records it was possible to determine that iberiotoxin binds to Ca(2+)-activate K+ channel in a bimolecular reaction. When the solution bathing the membrane are 300 mM K+ internal and 300 mM Na+ external the toxin second order association rate constant is 3.3 x 10(6) s-1 M-1 and the first order dissociation rate constant is 3.8 x 10(-3) s-1, yielding an apparent equilibrium dissociation constant of 1.16 nM. This constant is 10-fold lower than that of charybdotoxin, and the values for the rate constants showed above indicate that this is mainly due to the very low dissociation rate constant; mean blocked time approximately 5 min. The fact that tetraethylammonium competitively inhibits the iberiotoxin binding to the channel is a strong suggestion that this toxin binds to the channel external vestibule. Increasing the external K+ concentration makes the association rate constant to decrease with no effect on the dissociation reaction indicating that the surface charges located in the external channel vestibule play an important role in modulating toxin binding.     

Natural bile acids and synthetic analogues modulate large conductance Ca2+-activated K+ (BKCa) channel activity in smooth muscle cells

Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid-induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BK(Ca) channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid-induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BK(Ca) channel activity is not due to Ca(2+)-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid-induced increase in BK(Ca) channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid-induced increases in BK(Ca) channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BK(Ca) channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BK(Ca) channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BK(Ca) channel activity may explain their relaxing effect on smooth muscle.     

Effects of redox agents on the Ca2+-activated K+ channel

The sensitivity to Ca2+ of the Ca2+-dependent K+ channel can be increased by the artificial electron donor system ascorbate + phenazine-methosulphate in a variety of animal cells. In the human erythrocyte the shift from the 'low' to the 'high-affinity' state seems to depend on the reduction of a membrane component accepting 2 electrons and with an standard redox potential (pH 7.5) of about 47 mV. The relevance of this redox modulation under physiological circumstances is unknown at the moment.     

Identification and characterization of a novel, shorter isoform of the small conductance Ca2+ -activated K+ channel SK2

Throughout the CNS, small conductance Ca(2+)-activated potassium (SK) channels modulate firing frequency and neuronal excitability. We have identified a novel, shorter isoform of standard SK2 (SK2-std) in mouse brain which we named SK2-sh. SK2-sh is alternatively spliced at exon 3 and therefore lacks 140 amino acids, which include transmembrane domains S3, S4 and S5, compared with SK2-std. Western blot analysis of mouse hippocampal tissue revealed a 47 kDa protein product as predicted for SK2-sh along with a 64 kDa band representing the standard SK2 isoform. Electrophysiological recordings from transiently expressed SK2-sh revealed no functional channel activity or interaction with SK2-std. With the help of real-time PCR, we found significantly higher expression levels of SK2-sh mRNA in cortical tissue from AD cases when compared with age-matched controls. A similar increase in SK2-sh expression was induced in cortical neurons from mice by cytokine exposure. Substantial clinical evidence suggests that excess cytokines are centrally involved in the pathogenesis of Alzheimer's disease. Thus, SK2-sh as a downstream target of cytokines, provide a promising target for additional investigation regarding potential therapeutic intervention.     

Role of the NH2 terminus in the assembly and trafficking of the intermediate conductance Ca2+-activated K+ channel hIK1

The role of the NH(2)-terminal leucine zipper and dileucine motifs of hIK1 in the assembly, trafficking, and function of the channel was investigated using cell surface immunoprecipitation, co-immunoprecipitation (Co-IP), immunoblot, and whole-cell patch clamp techniques. Mutation of the NH(2)-terminal leucine zipper at amino acid positions 18 and 25 (L18A/L25A) resulted in a complete loss of steady-state protein expression, cell surface expression, and whole-cell current density. Inhibition of proteasomal degradation with lactacystin restored L18A/L25A protein expression, although this channel was not expressed at the cell surface as assessed by cell surface immunoprecipitation and whole-cell patch clamp. In contrast, inhibitors of lysosomal degradation (leupeptin/pepstatin) and endocytosis (chloroquine) had little effect on L18A/L25A protein expression or localization. Further studies confirmed the rapid degradation of this channel, having a time constant of 19.0 +/- 1.3 min compared with 3.2 +/- 0.8 h for wild type hIK1. Co-expression studies demonstrated that the L18A/L25A channel associates with wild type channel, thereby attenuating its expression at the cell surface. Co-IP studies confirmed this association. However, L18A/L25A channels failed to form homotetrameric channels, as assessed by Co-IP, suggesting the NH(2) terminus plays a role in tetrameric channel assembly. As with the leucine zipper, mutation of the dileucine motif to alanines, L18A/L19A, resulted in a near complete loss in steady-state protein expression with the protein being similarly targeted to the proteasome for degradation. In contrast to our results on the leucine zipper, however, both chloroquine and growing the cells at the permissive temperature of 27 degrees C restored expression of L18A/L19A at the cell surface, suggesting that the defect in the channel trafficking is the result of a subtle folding error. In conclusion, we demonstrate that the NH(2) terminus of hIK1 contains overlapping leucine zipper and dileucine motifs essential for channel assembly and trafficking to the plasma membrane.     

Structural determinants for functional coupling between the beta and alpha subunits in the Ca2+-activated K+ (BK) channel

High conductance, calcium- and voltage-activated potassium (BK, MaxiK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (beta) subunits. The most remarkable effects of beta1 and beta2 subunits are an increase of the calcium sensitivity and the slow down of channel kinetics. However, the detailed characteristics of channels formed by alpha and beta1 or beta2 are dissimilar, the most remarkable difference being a reduction of the voltage sensitivity in the presence of beta1 but not beta2. Here we reveal the molecular regions in these beta subunits that determine their differential functional coupling with the pore-forming alpha-subunit. We made chimeric constructs between beta1 and beta2 subunits, and BK channels formed by alpha and chimeric beta subunits were expressed in Xenopus laevis oocytes. The electrophysiological characteristics of the resulting channels were determined using the patch clamp technique. Chimeric exchange of the different regions of the beta1 and beta2 subunits demonstrates that the NH3 and COOH termini are the most relevant regions in defining the behavior of either subunit. This strongly suggests that the intracellular domains are crucial for the fine tuning of the effects of these beta subunits. Moreover, the intracellular domains of beta1 are responsible for the reduction of the BK channel voltage dependence. This agrees with previous studies that suggested the intracellular regions of the alpha-subunit to be the target of the modulation by the beta1-subunit.     

pH effects on high conductance Ca2+-activated K+ channels (BK(Ca)) in human internal mammary artery smooth muscle cells

INTRODUCTION: In vascular smooth muscle cells, different types of K+ channels participate in the regulation of membrane potential and consequently in the contractile behavior of the vessel. There is little information about the properties and role of K+ channels in human internal mammary artery (HIMA), the vessel of choice for coronary revascularization. METHODS: Patch-clamp technique on isolated HIMA smooth muscle cells was used. RESULTS: This work presents for the first time single-channel properties of the high conductance Ca2+-activated K+ channel (BK(Ca)) of HIMA. It presents a single-channel conductance of 228+/-4 pS (n=44, 8 cells), is sensitive to 100 nM iberiotoxin, and its open probability is Ca2+- and voltage-dependent. Inside-out results show that BK(Ca) channels in HIMA are directly activated by increasing the pH of intracellular media (NPo=0.096+/-0.032 at pH 7.4 and NPo=0.459+/-0.111 at pH 7.6, n=12 cells, p<0.05) and inhibited by lowering this pH (NPo=0.175+/-0.067 at pH 7.4 and NPo=0.051+/-0.019 at pH 6.8, n=13 cells, p<0.05). CONCLUSIONS: The evidences presented about single-channel properties and intracellular pH sensitivity of BK(Ca) from HIMA smooth muscle cells provide useful information to elucidate physiological or pathological mechanisms in this vessel, as well as for future studies where drugs could have BK(Ca) channels as targets for pharmacological therapies.     

Wanderlust kinetics and variable Ca(2+)-sensitivity of Drosophila, a large conductance Ca(2+)-activated K+ channel, expressed in oocytes.

Cloned large conductance Ca(2+)-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5 mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels.     

Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.     

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.