An amino acid outside the pore region influences apamin sensitivity in small conductance Ca2+-activated K+ channels

Small conductance calcium-activated potassium channels (SK, K(Ca)) are a family of voltage-independent K+ channels with a distinct physiology and pharmacology. The bee venom toxin apamin inhibits exclusively the three cloned SK channel subtypes (SK1, SK2, and SK3) with different affinity, highest for SK2, lowest for SK1, and intermediate for SK3 channels. The high selectivity of apamin made it a valuable tool to study the molecular makeup and function of native SK channels. Three amino acids located in the outer vestibule of the pore are of particular importance for the different apamin sensitivities of SK channels. Chimeric SK1 channels, enabling the homomeric expression of the rat SK1 (rSK1) subunit and containing the core domain (S1-S6) of rSK1, are apamin-insensitive. By contrast, channels formed by the human orthologue human SK1 (hSK1) are sensitive to apamin. This finding hinted at the involvement of regions beyond the pore as determinants of apamin sensitivity, because hSK1 and rSK1 have an identical amino acid sequence in the pore region. Here we investigated which parts of the channels outside the pore region are important for apamin sensitivity by constructing chimeras between apamin-insensitive and -sensitive SK channel subunits and by introducing point mutations. We demonstrate that a single amino acid situated in the extracellular loop between the transmembrane segments S3 and S4 has a major impact on apamin sensitivity. Our findings enabled us to convert the hSK1 channel into a channel that was as sensitive for apamin as SK2, the SK channel with the highest sensitivity.     

Inhibition of red cell Ca2+-dependent K+ channels by snake venoms

We have investigated the effects of several snake venoms on the Ca2+-dependent K+ channels of human red cells. A heat-resistant component of the venom of the snake Notechis scutatus irreversibly inhibited Ca2+-dependent K+ transport with a Ki value of 0.1-0.2 micrograms/ml. Metabolic changes of the cells modified the maximal effect of the venom. Binding of the venom required extracellular Ca2+ and was quick, but development of full inhibition required additional time. The effects of the venoms from Notechis scutatus and Leiurus quinquestriatus were additive, suggesting that both venoms act through different mechanisms. Venoms of the snakes Vipera russelli russelli and Oxyuranus scutellatus also inhibited Ca2+-dependent K+ transport with the same characteristics as the Notechis scutatus venom.     

Apamin: a specific toxin to study a class of Ca2+-dependent K+ channels

Apamin is a bee venom neurotoxin of 18 amino-acids containing two disulfide bridges. Current clamp and voltage clamp experiments have shown that externally applied apamin blocks specifically at low concentration (0.1 microM) the Ca2+-dependent slow K+ conductance which mediates the long-lasting after-hyperpolarization in neuroblastoma cells and rat muscle cells in culture. The apamin-sensitive Ca2+-dependent slow K+ conductance is voltage-dependent and tetraethylammonium (TEA) insensitive. It is distinct from the high conductance Ca2+-dependent K+ channel revealed by patch clamp experiments. Biochemical characterization of the apamin receptor in rat striated muscle, neuroblastoma cells, rat synaptosomes, smooth muscles and hepatocytes was carried out with the use of a radiolabelled monoiodo-apamin derivative (125I-apamin) of high specific radioactivity (2 000 Ci/mmol). The dissociation constant of the apamin-receptor complex is between 15 and 60 pM for all tissue preparations. The density of binding sites is very low; it varied between 1 and 40 fmol/mg of protein. Radiation inactivation analysis indicates a molecular weight for the apamin receptor of 250 000 daltons whereas affinity labelling with 125I-apamin results in covalent labelling of a single polypeptide chain with a molecular weight of about 30 000 daltons. We conclude that the apamin-sensitive Ca2+-dependent K+ channel is probably a large oligomeric structure containing one subunit of 30 000 daltons.     

Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength

Charybdotoxin (CTX), a small, basic protein from scorpion venom, strongly inhibits the conduction of K ions through high-conductance, Ca2+-activated K+ channels. The interaction of CTX with Ca2+-activated K+ channels from rat skeletal muscle plasma membranes was studied by inserting single channels into uncharged planar phospholipid bilayers. CTX blocks K+ conduction by binding to the external side of the channel, with an apparent dissociation constant of approximately 10 nM at physiological ionic strength. The dwell-time distributions of both blocked and unblocked states are single-exponential. The toxin association rate varies linearly with the CTX concentration, and the dissociation rate is independent of it. CTX is competent to block both open and closed channels; the association rate is sevenfold faster for the open channel, while the dissociation rate is the same for both channel conformations. Membrane depolarization enhances the CTX dissociation rate e-fold/28 mV; if the channel's open probability is maintained constant as voltage varies, then the toxin association rate is voltage independent. Increasing the external solution ionic strength from 20 to 300 mM (with K+, Na+, or arginine+) reduces the association rate by two orders of magnitude, with little effect on the dissociation rate. We conclude that CTX binding to the Ca2+-activated K+ channel is a bimolecular process, and that the CTX interaction senses both voltage and the channel's conformational state. We further propose that a region of fixed negative charge exists near the channel's CTX-binding site.     

Mechanisms underlying the nitric oxide inhibitory effects in mouse ileal longitudinal muscle

We investigated the mechanisms involved in the nitric oxide (NO)-induced inhibitory effects on longitudinal smooth muscle of mouse ileum, using organ bath technique. Exogenously applied NO, delivered as sodium nitroprusside (SNP; 0.1-100 micromol/L) induced a concentration-dependent reduction of the ileal spontaneous contractions. 1H-[1,2,4]oxadiazolol[4,3,a]quinoxalin-1-one (ODQ; 1 micromol/L), a guanilyl cyclase inhibitor, reduced the SNP-induced effects. Tetraethylammonium chloride (20 mmol/L), a non-selective K+ channel blocker, and charybdotoxin (0.1 micromol/L), blocker of large conductance Ca2+-dependent K+ channels, significantly reduced SNP-induced inhibitory effects. In contrast, apamin (0.1 micromol/L), blocker of small conductance Ca2+-dependent K+ channels, was not able to affect the response to SNP. Ciclopiazonic acid (10 micromol/L) or thapsigargin (0.1 micromol/L), sarcoplasmatic reticulum Ca2+-ATPase inhibitors, decreased the SNP-inhibitory effects. Ryanodine (10 micromol/L), inhibitor of Ca2+ release from ryanodine-sensitive intracellular stores, significantly reduced the SNP inhibitory effects. The membrane permeable analogue of cGMP, 8-bromoguanosine 3',5'-cyclic monophosphate (100 micromol/L), also reduced spontaneous mechanical activity, and its effect was antagonized by ryanodine. The present study suggests that NO causes inhibitory effects on longitudinal smooth muscle of mouse ileum through cGMP which in turn would activate the large conductance Ca2+-dependent K+ channels, via localized ryanodine-sensitive Ca2+ release.     

Mitogen-induced changes in Ca2+ permeability are not mediated by voltage-gated K+ channels

Binding of mitogenic lectins to T lymphocytes results in elevated cytoplasmic Ca2+ concentrations ([Ca2+]i). This change in [Ca2+]i is thought to be essential for cellular proliferation. In addition, the lectins increase the conductance to K+ through voltage-sensitive channels. Based on the inhibitory effect of K+ channel blockers on lectin-induced mitogenesis, it has been suggested that Ca2+ could enter the cells through these activated K+ channels (Chandy, K. G., De Coursey, T. E., Cahalan, M. D., McLaughlin, C., and Gupta, S. (1984) J. Exp. Med. 160, 369-385; Chandy, K. G., De Coursey, T. E., Cahalan, M. D., and Gupta, S. (1985) J. Clin. Immunol. 5, 1-5). This hypothesis was tested experimentally by measuring the effect of activation or blockade of K+ channels on [Ca2+]i using quin-2 and indo-1 and by determining the effect of K+ channel blockers on lectin-induced proliferation. We found that: depolarization of the membrane, which is expected to open the K+ channels, failed to increase [Ca2+]i, K+ channel blockers such as tetraethylammonium and 4-aminopyridine had only a marginal effect on the lectin-induced increase in [Ca2+]i, and the inhibitory effect of K+ channel blockers on proliferation was found to be nonspecific, occurring also when proliferation was triggered by phorbol esters under conditions where [Ca2+]i is not elevated. It is concluded that the lectin-induced changes in [Ca2+]i are not mediated by the opening of voltage-gated K+ channels.     

A peptide derived from the Shaker B K+ channel produces short and long blocks of reconstituted Ca(2+)-dependent K+ channels.

A 20 amino acid synthetic peptide, corresponding to the amino-terminal region of the Shaker B (ShB) K+ channel and responsible for its fast inactivation, can block large conductance Ca(2+)-dependent K+ channels from rat brain and muscle. The ShB inactivation peptide produces two kinetically distinct blocking events in these channels. At lower concentrations, it produces short blocks, and at higher concentrations long-lived blocks also appear. The L7E mutant peptide produces only infrequent short blocks (no long-lived blocks) at a much higher concentration. Internal tetraethylammonium competes with the peptide for the short block, which is also relieved by K+ influx. These results suggest that the peptide induces the short block by binding within the pore of Ca(2+)-dependent K+ channels. The long block is not affected by increased K+ influx, indicating that the binding site mediating this block may be different from that involved in the short block. The short block of Ca(2+)-dependent K+ channels and the inactivation of Shaker exhibit similar characteristics with respect to blocking affinity and open pore blockade. This suggests a conserved binding region for the peptide in the pore regions of these very different classes of K+ channel.     

Identification and characterization of stretch-activated ion channels in pollen protoplasts

Pollen tube growth requires a Ca2+ gradient, with elevated levels of cytosolic Ca2+ at the growing tip. This gradient's magnitude oscillates with growth oscillation but is always maintained. Ca2+ influx into the growing tip is necessary, and its magnitude also oscillates with growth. It has been widely assumed that stretch-activated Ca2+ channels underlie this influx, but such channels have never been reported in either pollen grains or pollen tubes. We have identified and characterized stretch-activated Ca2+ channels from Lilium longiflorum pollen grain and tube tip protoplasts. The channels were localized to a small region of the grain protoplasts associated with the site of tube germination. In addition, we find a stretch-activated K+ channel as well as a spontaneous K+ channel distributed over the entire grain surface, but neither was present at the germination site or at the tip. Neither stretch-activated channel was detected in the grain protoplasts unless the grains were left in germination medium for at least 1 h before protoplast preparation. The stretch-activated channels were inhibited by a spider venom that is known to block stretch-activated channels in animal cells, but the spontaneous channel was unaffected by the venom. The venom also stopped pollen tube germination and elongation and blocked Ca2+ entry into the growing tip, suggesting that channel function is necessary for growth.     

Vasoactive intestinal peptide activates Ca2(+)-dependent K+ channels through a cAMP pathway in mouse lacrimal cells

The action of vasoactive intestinal peptide (VIP) on Ca2(+)-dependent K+ currents, in dissociated mouse lacrimal cells, was investigated using patch clamp techniques. In whole cell recordings, VIP (10-100 pM) increased the magnitude of the Ca2(+)-dependent K+ current. In single channel recordings, VIP increased the fraction of time the large charybdotoxin-sensitive Ca2(+)-activated K+ channel spent in the open state. The activity of this channel was also increased by adding forskolin or 8-bromo cAMP to the bath. Additionally, application of either cAMP or catalytic subunit of cAMP-dependent protein kinase directly to the cytoplasmic surface of excised inside out patches reversibly lengthened the time Ca2(+)-activated K+ channels spent in the open state. These data suggest that VIP stimulates Ca2(+)-activated K+ channels by a cAMP-dependent pathway in mouse lacrimal acinar cells.     

High affinity inhibition of Ca(2+)-dependent K+ channels by cytochrome P-450 inhibitors.

The Ca(2+)-dependent K+ channel of human red cells was inhibited with high affinity by several imidazole antimycotics which are potent inhibitors of cytochrome P-450. IC50 values were (in microM): clotrimazole, 0.05; tioconazole, 0.3; miconazole, 1.5; econazole, 1.8. Inhibition of the channel was also found with other drugs with known cytochrome P-450 inhibitory effect. However, no inhibition was obtained with carbon monoxide (CO). This suggests that, given the high selectivity of the above inhibitors for the heme moiety, a different but closely related to cytochrome P-450 kind of hemoprotein may be involved in the regulation of the red cell Ca(2+)-dependent K+ channel. Clotrimazole also inhibited two other charybdotoxin-sensitive Ca(2+)-dependent K+ channels, those of rat thymocytes (IC50 = 0.1-0.2 microM) and of Ehrlich ascites tumor cells (IC50 = 0.5 microM). Imidazole antimycotics inhibit also receptor-operated Ca2+ channels (Montero, M., Alvarez, J. and García-Sancho, J. (1991) Biochem. J. 277, 73-79). This suggests that both Ca2+ and Ca(2+)-dependent K+ channels might have a similar regulatory mechanism involving a cytochrome.     

Kinetic differences between Ca(2+)-dependent K+ channels in smooth muscle cells isolated from normal and atherosclerotic human aorta.

The properties of large conductance Ca(2+)-dependent K+ channels in smooth muscle cells (SMC) isolated from normal and atherosclerotic human aorta were studied using the patch-clamp technique. It was shown that SMC from normal human aorta possess a homogeneous population of normal Ca(2+)-dependent K+ channels. In atherosclerotic aorta two kinetically different types of these channels could be distinguished: along with normal 'long' (L)-type channels there appeared channels of 'short' (s)-type. Under similar conditions s-type channels had about a four times shorter mean open time. About five times higher [Ca2+]in was necessary for s-type channels to reach the probability of the channels being open equal to L-type channels. No differences in conductance and voltage dependency were found between the two channel types. Channels of the s-type resembled those previously described in SMC isolated from foetal human aorta. Thus, it can be suggested that during the development of atherosclerosis a population of SMC with s-type Ca(2+)-dependent K+ channels appears in human aorta.     

Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage

Large conductance Ca2+-activated K+ (BK) channels belong to the S4 superfamily of K+ channels that include voltage-dependent K+ (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH2-terminus. The BK channel is activated by internal Ca2+, and using chimeric channels and mutagenesis, three distinct Ca2+-dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca2+-binding domains activate the BK channel when cytoplasmic Ca2+ reaches micromolar concentrations, and a low Ca2+ affinity mechanism may be involved in the physiological regulation by Mg2+. The presence in the BK channel of multiple Ca2+-binding sites explains the huge Ca2+ concentration range (0.1 microM-100 microM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca2+. Therefore, Ca2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.     

Ca2+-dependent K+ transport in lymphocytes

The treatment of rat thymocytes with A23187 + Ca2+, ascorbate-phenazine methosulphate or propranolol induced quinine-sensitive fluxes of K+ (Rb+) suggesting the presence in the cell membrane of Ca2+-dependent K+ channels. Concanavalin A induced K+ channel activation only at very high doses (13 micrograms/ml). Neither quinine nor the increase of the K+ concentration in the medium to 30 mM prevented the stimulation of amino acid transport induced by concanavalin A, suggesting that the Ca2+-dependent K+ channel is not involved in the early phenomena of lymphocyte activation.     

Activation of a small-conductance Ca(2+)-dependent K+ channel contributes to bradykinin-induced stimulation of nitric oxide synthesis in pig aortic endothelial cells.

Bradykinin-induced K+ currents, membrane hyperpolarization, as well as rises in cytoplasmic Ca2+ and cGMP levels were studied in endothelial cells cultured from pig aorta. Exposure of endothelial cells to 1 microM bradykinin induced a whole-cell K+ current and activated a small-conductance (approximately 9 pS) K+ channel in on-cell patches. This K+ channel lacked voltage sensitivity, was activated by increasing the Ca2+ concentration at the cytoplasmic face of inside-out patches and blocked by extracellular tetrabutylammonium (TBA). Bradykinin concomitantly increased membrane potential and cytoplasmic Ca2+ of endothelial cells. In high (140 mM) extracellular K+ solution, as well as in the presence of the K(+)-channel blocker TBA (10 mM), bradykinin-induced membrane hyperpolarization was abolished and increases in cytoplasmic Ca2+ were reduced to a slight transient response. Bradykinin-induced rises in intracellular cGMP levels which reflect Ca(2+)-dependent formation of EDRF(NO) were clearly attenuated in the presence of TBA (10 mM). Our results suggest that bradykinin hyperpolarizes pig aortic endothelial cells by activation of small-conductance Ca(2+)-activated K+ channels. Opening of these K+ channels results in membrane hyperpolarization which promotes Ca2+ entry, and consequently, NO synthesis.     

Developmental differences in Ca2+-activated K+ channel activity in ovine basilar artery

A primary determinant of vascular smooth muscle (VSM) tone and contractility is the resting membrane potential, which, in turn, is influenced heavily by K+ channel activity. Previous studies from our laboratory and others have demonstrated differences in the contractility of cerebral arteries from near-term fetal and adult animals. To test the hypothesis that these contractility differences result from maturational changes in voltage-gated K+ channel function, we compared this function in VSM myocytes from adult and fetal sheep cerebral arteries. The primary current-carrying, voltage-gated K+ channels in VSM myocytes are the large conductance Ca2+-activated K+ channels (BKCa) and voltage-activated K+ (KV) channels. We observed that at voltage-clamped membrane potentials of +60 mV in perforated whole cell studies, the normalized outward current densities in fetal myocytes were >30% higher than in those of the adult (P < 0.05) and that these were predominantly due to iberiotoxin-sensitive currents from BKCa channels. Excised, insideout membrane patches revealed nearly identical unitary conductances and Hill coefficients for BKCa channels. The plot of log intracellular [Ca2+] ([Ca2+]i) versus voltage for half-maximal activation (V(1/2)) yielded linear and parallel relationships, and the change in V(1/2) for a 10-fold change in [Ca2+] was also similar. Channel activity increased e-fold for a 19 +/- 2-mV depolarization for adult myocytes and for an 18 +/- 1-mV depolarization for fetal myocytes (P > 0.05). However, the relationship between BKCa open probability and membrane potential had a relative leftward shift for the fetal compared with adult myocytes at different [Ca2+]i. The [Ca2+] for half-maximal activation (i.e., the calcium set points) at 0 mV were 8.8 and 4.7 microM for adult and fetal myocytes, respectively. Thus the increased BKCa current density in fetal myocytes appears to result from a lower calcium set point.     

Inhibition of chloroplast ATPase by the K+ channel blocker alpha-dendrotoxin.

Possible structural and functional similarities between the channel part, CF0, of chloroplast ATPase (CF0CF1) and ion channels permeable to monovalent cations were investigated using high-affinity toxins mainly targeted against voltage-sensitive K+ channels. In particular, the effect of the K(+)-channel blocker alpha-dendrotoxin and the crude scorpion venom of Leiurus quinquestriatus hebraeus (LQ venom) on ATP synthesis in thylakoid membranes and in CF0CF1-containing liposomes was characterised. Alpha-dendrotoxin (K(i) approximately 5.05 microM) and the LQ venom (K(i) approximately 1.55 micrograms/ml) specifically inhibited ATP synthesis in thylakoid membranes and in CF0CF1-containing liposomes. Our results show that alpha-dendrotoxin and peptides of the LQ venom with an apparent molecular mass of about 4.0 kDa, probably isoforms of charybdotoxin, specifically bind to CF0CF1. This binding was reversible and induced a high leak conductance for H+ through CF0. The Ca(2+)-dependent ATPase activity of the isolated soluble part of CF0CF1 (CF1) was completely inhibited by 1 microM alpha-dendrotoxin, while the crude LQ venom, at concentrations up to 10 micrograms/ml, had no affect on ATPase activity. The concentration dependence of the inhibition by alpha-dendrotoxin indicates that approximately 2 mol alpha-dendrotoxin bind/mol CF0CF1 and 1 mol alpha-dendrotoxin/mol CF1. Known inhibitors of H(+)-flow-through CF0 acted in the presence of alpha-dendrotoxin synergistically. Dicyclohexylcarbodiimide and venturicidin, in contrast to their known effect of blocking H(+)-flow-through CF0, increased the leak conductance through CF0 in the presence of alpha-dendrotoxin drastically. This uncoupling effect indicates that their normal mode of blocking is a secondary effect. Binding of the inhibitors to their respective sites apparently does not affect the proton pathway in CF0, but induces a conformation which closes the channel part for H+. Protein sequence comparison between the known binding site of charybdotoxin in the shaker K+ channel from Drosophila [MacKinnon, R. & Heginbotham, L. (1990) Neuron 5, 767-771] and the choroplast ATPase showed that subunit III reveals a significant similarity (64%) in parts of its sequence (Gln28-Leu53) to the helix 5 and helix 6 (S5-S6) linker region (Ala413-Cys462; the charybdotoxin-binding site) of the shaker K+ channel. According to secondary-structure predictions, the homologous sequences in subunit III and the shaker K+ channel represent putative hydrophilic loops connecting two transmembrane alpha-helices. Apparently the shaker K+ channel and subunit III share significant topological features in these hydrophilic loops which may be part of the respective channel entrance.     

Ion selectivity of porcine skeletal muscle Ca2+ release channels is unaffected by the Arg615 to Cys615 mutation.

The Arg615 to Cys615 mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of malignant hyperthermia susceptible (MHS) pigs results in a decreased sensitivity of the channel to inhibitory Ca2+ concentrations. To investigate whether this mutation also affects the ion selectivity filter of the channel, the monovalent cation conductances and ion permeability ratios of single Ca2+ release channels incorporated into planar lipid bilayers were compared. Monovalent cation conductances in symmetrical solutions were: Li+, 183 pS +/- 3 (n = 21); Na+, 474 pS +/- 6 (n = 29); K+, 771 pS +/- 7 (n = 29); Rb+, 502 pS +/- 10 (n = 22); and Cs+, 527 pS +/- 5 (n = 16). The single-channel conductances of MHS and normal Ca2+ release channel were not significantly different for any of the monovalent cations tested. Permeability ratios measured under biionic conditions had the permeability sequence Ca2+ >> Li+ > Na+ > K+ > or Rb+ > Cs+, with no significant difference noted between MHS and normal channels. This systematic examination of the conduction properties of the pig skeletal muscle Ca2+ release channel indicated a higher Ca2+ selectivity (PCa2+:Pk+ approximately 15.5) than the sixfold Ca2+ selectivity previously reported for rabbit skeletal (Smith et al., 1988) or sheep cardiac muscle (Tinker et al., 1992) Ca2+ release channels. These results also indicate that although Ca2+ regulation of Ca2+ release channel activity is altered, the Arg615 to Cys615 mutation of the porcine Ca2+ release channel does not affect the conductance or ion selectivity properties of the channel.     

Contryphan-Vn: a modulator of Ca2+-dependent K+ channels

Contryphan-Vn is a D-tryptophan-containing disulfide-constrained nonapeptide isolated from the venom of Conus ventricosus, the single Mediterranean cone snail species. The structure of the synthetic Contryphan-Vn has been determined by NMR spectroscopy. Unique among Contryphans, Contryphan-Vn displays the peculiar presence of a Lys-Trp dyad, reminiscent of that observed in several voltage-gated K(+) channel blockers. Electrophysiological experiments carried out on dorsal unpaired median neurons isolated from the cockroach (Periplaneta americana) nerve cord on rat fetal chromaffin cells indicate that Contryphan-Vn affects both voltage-gated and Ca(2+)-dependent K(+) channel activities, with composite and diversified effects in invertebrate and vertebrate systems. Voltage-gated and Ca(2+)-dependent K(+) channels represent the first functional target identified for a conopeptide of the Contryphan family. Furthermore, Contryphan-Vn is the first conopeptide known to modulate the activity of Ca(2+)-dependent K(+) channels.     

Beta 1-subunits are required for regulation of coupling between Ca2+ transients and Ca2+-activated K+ (BK) channels by protein kinase C

Colonic myocytes have spontaneous, localized, Ins (1,4,5) trisphosphate (IP3) receptor-dependent Ca2+ transients that couple to the activation of Ca2+-dependent K+ channels and spontaneous transient outward currents (STOCs). We previously reported that the coupling strength between spontaneous Ca2+ transients and large conductance Ca2+ activated K+ (BK) channels is regulated by Ca2+ influx through nonselective cation channels and activation of protein kinase C (PKC). Here, we used confocal microscopy and the patch-clamp technique to further investigate the coupling between localized Ca2+ transients and STOCs in colonic myocytes from animals lacking the regulatory beta1-subunit of BK channels. Myocytes from beta1-knockout (beta1-/-) animals loaded with fluo 4 showed typical localized Ca2+ transients, but the STOCs coupled to these events were of abnormally low amplitude. Reduction in external Ca2+ or application of inhibitors of nonselective cation channels (SKF-96365) caused no significant change in the amplitude or frequency of STOCs. Likewise, an inhibitor of PKC, GF 109203X, had no significant effect on STOCs. Single-channel recording from BK channels showed that application of an activator (PMA) and an inhibitor (GF 109203X) of PKC did not affect BK channel openings in myocytes of beta1-/- mice. These data show that PKC-dependent regulation of coupling strength between Ca2+ transients and STOCs in colonic myocytes depends upon the interaction between alpha- and beta1-subunits.