Selectivity filter gating in large-conductance Ca(2+)-activated K+ channels

Membrane voltage controls the passage of ions through voltage-gated K (K(v)) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of K(v) channels is well established, it is not clear if such a cytoplasmic gate exists in all K(+) channels. Some studies on large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that BK channels are gated by the pore selectivity filter. If the BK channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. Thus, we tested tetrabutyl ammonium (TBA) and the Shaker "ball" peptide (BP) on BK channels with either K(+) or Rb(+) as the permeant ion. When tested in K(+) solutions, both TBA and the BP acted as open-channel blockers of BK channels, and the BP interfered with channel closing. In contrast, when Rb(+) replaced K(+) as the permeant ion, TBA and the BP blocked both closed and open BK channels, and the BP no longer interfered with channel closing. We also tested the cytoplasmically gated Shaker K channels and found the opposite behavior: the interactions of TBA and the BP with these K(v) channels were independent of the permeant ion. Our results add significantly to the evidence against a cytoplasmic gate in BK channels and represent a positive test for selectivity filter gating.     

Mitochondrial Ca2+-activated K+ channels more efficiently reduce mitochondrial Ca2+ overload in rat ventricular myocytes

We investigated the role of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel, the mitochondrial big-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, and the mitochondrial permeability transition pore (MPTP) in the ouabain-induced increase of mitochondrial Ca(2+) in native rat ventricular myocytes by loading cells with rhod 2-AM. To overload mitochondrial Ca(2+), we pretreated cells with ouabain before applying mitochondrial K(ATP) or BK(Ca) channel and/or MPTP opener. Ouabain (1 mM) increased the rhod 2-sensitive fluorescence intensity (160 +/- 5.0% of control), which was dramatically decreased to the control level on application of diazoxide and NS-1619 in a dose-dependent manner (half-inhibition concentrations of 78.3 and 7.78 muM for diazoxide and NS-1619, respectively). This effect was reversed by selective inhibition of the mitochondrial K(ATP) channel by 5-hydroxydecanoate, the mitochondrial BK(Ca) channel by paxilline, and the MPTP by cyclosporin A. Although diazoxide did not efficiently reduce mitochondrial Ca(2+) during prolonged exposure to ouabain, NS-1619 reduced mitochondrial Ca(2+). These results suggest that although mitochondrial BK(Ca) and K(ATP) channels contribute to reduction of ouabain-induced mitochondrial Ca(2+) overload, activation of the mitochondrial BK(Ca) channel more efficiently reduces ouabain-induced mitochondrial Ca(2+) overload in our experimental model.     

BmBKTx1, a novel Ca2+-activated K+ channel blocker purified from the Asian scorpion Buthus martensi Karsch

BmBKTx1 is a novel short chain toxin purified from the venom of the Asian scorpion Buthus martensi Karsch. It is composed of 31 residues and is structurally related to SK toxins. However, when tested on the cloned rat SK2 channel, it only partially inhibited rSK2 currents, even at a concentration of 1 microm. To screen for other possible targets, BmBKTx1 was then tested on isolated metathoracic dorsal unpaired median neurons of Locusta migratoria, in which a wide variety of ion channels are expressed. The results suggested that BmBKTx1 could specifically block voltage-gated Ca(2+)-activated K(+) currents (BK-type). This was confirmed by testing the BmBKTx1 effect on the alpha subunits of BK channels of the cockroach (pSlo), fruit fly (dSlo), and human (hSlo), heterologously expressed in HEK293 cells. The IC(50) for channel blocking by BmBKTx1 was 82 nm for pSlo and 194 nm for dSlo. Interestingly, BmBKTx1 hardly affected hSlo currents, even at concentrations as high as 10 microm, suggesting that the toxin might be insect specific. In contrast to most other scorpion BK blockers that also act on the Kv1.3 channel, BmBKTx1 did not affect this channel as well as other Kv channels. These results show that BmBKTx1 is a novel kind of blocker of BK-type Ca(2+)-activated K(+) channels. As the first reported toxin active on the Drosophila Slo channel dSlo, it will also greatly facilitate studying the physiological role of BK channels in this model organism.     

Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K(+) channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca(2+) -activated K(+) channels (BK(Ca)). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K(+) currents carried by BK(Ca) channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC(50) 324 μM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 μM) can also block whole-cell K(+) currents (~45% blockage) in which, under our working conditions, BK(Ca) is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK(Ca) channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.     

Hypotonicity-induced TRPV4 function in renal collecting duct cells: modulation by progressive cross-talk with Ca2+-activated K+ channels

The mouse cortical collecting duct (CCD) M-1 cells were grown to confluency on coverslips to assess the interaction between TRPV4 and Ca(2+)-activated K(+) channels. Immunocytochemistry demonstrated strong expression of TRPV4, along with the CCD marker, aquaporin-2, and the Ca(2+)-activated K(+) channels, the small conductance SK3 (K(Ca)2.3) channel and large conductance BKα channel (K(Ca)1.1). TRPV4 overexpression studies demonstrated little physical dependency of the K(+) channels on TRPV4. However, activation of TRPV4 by hypotonic swelling (or GSK1016790A, a selective agonist) or inhibition by the selective antagonist, HC-067047, demonstrated a strong dependency of SK3 and BK-α activation on TRPV4-mediated Ca(2+) influx. Selective inhibition of BK-α channel (Iberiotoxin) or SK3 channel (apamin), thereby depolarizing the cells, further revealed a significant dependency of TRPV4-mediated Ca(2+) influx on activation of both K(+) channels. It is concluded that a synergistic cross-talk exists between the TRPV4 channel and SK3 and BK-α channels to provide a tight functional regulation between the channel groups. This cross-talk may be progressive in nature where the initial TRPV4-mediated Ca(2+) influx would first activate the highly Ca(2+)-sensitive SK3 channel which, in turn, would lead to enhanced Ca(2+) influx and activation of the less Ca(2+)-sensitive BK channel.     

Characterization of voltage-and Ca2+-activated K+ channels in rat dorsal root ganglion neurons

Auxiliary beta-subunits associated with pore-forming Slo1 alpha-subunits play an essential role in regulating functional properties of large-conductance, voltage- and Ca(2+)-activated K(+) channels commonly termed BK channels. Even though both noninactivating and inactivating BK channels are thought to be regulated by beta-subunits (beta1, beta2, beta3, or beta4), the molecular determinants underlying inactivating BK channels in native cells have not been extensively demonstrated. In this study, rbeta2 (but not rbeta3-subunit) was identified as a molecular component in rat lumbar L4-6 dorsal root ganglia (DRG) by RT-PCR responsible for inactivating large-conductance Ca(2+)-dependent K(+) currents (BK(i) currents) in small sensory neurons. The properties of native BK(i) currents obtained from both whole-cell and inside-out patches are very similar to inactivating BK channels produced by co-expressing mSlo1 alpha- and hbeta2-subunits in Xenopus oocytes. Intracellular application of 0.5 mg/ml trypsin removes inactivation of BK(i) channels, and the specific blockers of BK channels, charybdotoxin (ChTX) and iberiotoxin (IbTX), inhibit these BK(i) currents. Single BK(i) channel currents derived from inside-out patches revealed that one BK(i) channel contained three rbeta2-subunits (on average), with a single-channel conductance about 217 pS under 160 K(+) symmetrical recording conditions. Blockade of BK(i) channels by 100 nM IbTX augmented firing frequency, broadened action potential waveform and reduced after-hyperpolarization. We propose that the BK(i) channels in small diameter DRG sensory neurons might play an important role in regulating nociceptive input to the central nervous system (CNS).     

The mitochondrial Ca2+-activated K+ channel activator, NS 1619 inhibits L-type Ca2+ channels in rat ventricular myocytes

We examined the effects of the mitochondrial Ca(2+)-activated K(+) (mitoBK(Ca)) channel activator NS 1619 on L-type Ca(2+) channels in rat ventricular myocytes. NS 1619 inhibited the Ca(2+) current in a dose-dependent manner. NS 1619 shifted the activation curve to more positive potentials, but did not have a significant effect on the inactivation curve. Pretreatment with inhibitors of membrane BK(Ca) channel, mitoBK(Ca) channel, protein kinase C, protein kinase A, and protein kinase G had little effect on the Ca(2+) current and did not alter the inhibitory effect of NS 1619 significantly. The application of additional NS 1619 in the presence of isoproterenol, a selective beta-adrenoreceptor agonist, reduced the Ca(2+) current to approximately the same level as a single application of NS 1619. In conclusion, our results suggest that NS 1619 inhibits the Ca(2+) current independent of the mitoBK(Ca) channel and protein kinases. Since NS 1619 is widely used to study mitoBK(Ca) channel function, it is essential to verify these unexpected effects of NS 1619 before experimental data can be interpreted accurately.     

SNAP-25 inhibits L-type Ca2+ channels in feline esophagus smooth muscle cells

We recently reported that non-secretory gastrointestinal smooth muscle cells also possessed SNARE proteins, of which SNAP-25 regulated Ca(2+)-activated (K(Ca)) and delayed rectifier K(+) channels (K(V)). Voltage-gated, long lasting (L-type) calcium channels (L(Ca)) play an important role in excitation-contraction coupling of smooth muscle. Here, we show that SNAP-25 could also directly inhibit the L-type Ca(2+) channels in feline esophageal smooth muscle cells at the SNARE complex binding synprint site. SNARE proteins could therefore regulate additional cell actions other than membrane fusion and secretion, in particular, coordinated muscle membrane excitability and contraction, through their actions on membrane Ca(2+) and K(+) channels.     

Interaction of SK(Ca) channels and L-type Ca(2+) channels in catecholamine secretion in the rat adrenal gland

We elucidated the interaction of small-conductance Ca(2+)-activated K(+) (SK(Ca)) channels and L-type Ca(2+) channels in muscarinic receptor-mediated control of catecholamine secretion in the isolated perfused rat adrenal gland. The muscarinic agonist methacholine (10-300 microM) produced concentration-dependent increases in adrenal output of epinephrine and norepinephrine. The SK(Ca) channel blocker apamin (1 microM) enhanced the methacholine-induced catecholamine responses. The facilitatory effect of apamin on the methacholine-induced catecholamine responses was not observed during treatment with the L-type Ca(2+) channel blocker nifedipine (3 microM) or Ca(2+)-free solution. Nifedipine did not affect the methacholine-induced catecholamine responses, but it inhibited the responses during treatment with apamin. The L-type Ca(2+) channel activator Bay k 8644 (1 microM) enhanced the methacholine-induced catecholamine responses, whereas the enhancement of the methacholine-induced epinephrine and norepinephrine responses were prevented and attenuated by apamin, respectively. These results suggest that SK(Ca) channels are activated by muscarinic receptor stimulation, which inhibits the opening of L-type Ca(2+) channels and thereby attenuates adrenal catecholamine secretion.     

Functional apical large conductance, Ca2+-activated, and voltage-dependent K+ channels are required for maintenance of airway surface liquid volume

Large conductance, Ca(2+)-activated, and voltage-dependent K(+) (BK) channels control a variety of physiological processes in nervous, muscular, and renal epithelial tissues. In bronchial airway epithelia, extracellular ATP-mediated, apical increases in intracellular Ca(2+) are important signals for ion movement through the apical membrane and regulation of water secretion. Although other, mainly basolaterally expressed K(+) channels are recognized as modulators of ion transport in airway epithelial cells, the role of BK in this process, especially as a regulator of airway surface liquid volume, has not been examined. Using patch clamp and Ussing chamber approaches, this study reveals that BK channels are present and functional at the apical membrane of airway epithelial cells. BK channels open in response to ATP stimulation at the apical membrane and allow K(+) flux to the airway surface liquid, whereas no functional BK channels were found basolaterally. Ion transport modeling supports the notion that apically expressed BK channels are part of an apical loop current, favoring apical Cl(-) efflux. Importantly, apical BK channels were found to be critical for the maintenance of adequate airway surface liquid volume because continuous inhibition of BK channels or knockdown of KCNMA1, the gene coding for the BK α subunit (KCNMA1), lead to airway surface dehydration and thus periciliary fluid height collapse revealed by low ciliary beat frequency that could be fully rescued by addition of apical fluid. Thus, apical BK channels play an important, previously unrecognized role in maintaining adequate airway surface hydration.     

Conducted vasoconstriction in rat mesenteric arterioles: role for dihydropyridine-insensitive Ca(2+) channels

The aim of this study was to evaluate the role of voltage-operated Ca(2+) channels in the initiation and conduction of vasoconstrictor responses to local micropipette electrical stimulation of rat mesenteric arterioles (28 +/- 1 microm, n = 79) in vivo. Local and conducted (600 microm upstream from the pipette) vasoconstriction was not blocked by TTX (1 micromol/l, n = 5), nifedipine, or nimodipine (10 micromol/l, n = 9). Increasing the K(+) concentration of the superfusate to 75 mmol/l did not evoke vasoconstriction, but this depolarizing stimulus reversibly abolished vasoconstrictor responses to current stimulation (n = 7). Addition of the T-type Ca(2+) antagonist mibefradil (10 micromol/l, n = 6) to the superfusate reversibly blocked local and conducted vasoconstriction to current stimulation. With the use of RT-PCR techniques, it was demonstrated that rat mesenteric arterioles <40 microm do not express mRNA for L-type Ca(2+) channels (alpha(1C)-subunit), whereas mRNA coding for T-type subunits was found (alpha(1G)- and alpha(1H)-subunits). The data indicate that L-type Ca(2+) channels are absent from rat mesenteric arterioles (<40 microm). Rather, the vasoconstrictor responses appear to rely on other types of voltage-gated, dihydropyridine-insensitive Ca(2+) channels, possibly of the T-type.     

Ca(2+)-regulated ion channels

Due to its high external and low internal concentration the Ca(2+) ion is used ubiquitously as an intracellular signaling molecule, and a great many Ca(2+)-sensing proteins have evolved to receive and propagate Ca(2+) signals. Among them are ion channel proteins, whose Ca(2+) sensitivity allows internal Ca(2+) to influence the electrical activity of cell membranes and to feedback-inhibit further Ca(2+) entry into the cytoplasm. In this review I will describe what is understood about the Ca(2+) sensing mechanisms of the three best studied classes of Ca(2+)-sensitive ion channels: Large-conductance Ca(2+)-activated K(+) channels, small-conductance Ca(2+)-activated K(+) channels, and voltage- gated Ca(2+) channels. Great strides in mechanistic understanding have be made for each of these channel types in just the past few years.     

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.     

Nonglutamate pore residues in ion selection and conduction in voltage-gated Ca(2+) channels

High-affinity, intrapore binding of Ca(2+) over competing ions is the essential feature in the ion selectivity mechanism of voltage-gated Ca(2+) channels. At the same time, several million Ca(2+) ions can travel each second through the pore of a single open Ca(2+) channel. How such high Ca(2+) flux is achieved in the face of tight Ca(2+) binding is a current area of inquiry, particularly from a structural point of view. The ion selectivity locus comprises four glutamate residues within the channel's pore. These glutamates make unequal contributions to Ca(2+) binding, underscoring a role for neighboring residues in pore function. By comparing two Ca(2+) channels (the L-type alpha(1C), and the non-L-type alpha(1A)) that differ in their pore properties but only differ at a single amino acid position near the selectivity locus, we have identified the amino-terminal neighbor of the glutamate residue in motif III as a determinant of pore function. This position is more important in the function of alpha(1C) channels than in alpha(1A) channels. For a systematic series of mutations at this pore position in alpha(1C), both unitary Ba(2+) conductance and Cd(2+) block of Ba(2+) current varied with residue volume. Pore mutations designed to make alpha(1C) more like alpha(1A) and vice versa revealed that relative selectivity for Ba(2+) over K(+) depended almost solely on pore sequence and not channel type. Analysis of thermodynamic mutant cycles indicates that the motif III neighbor normally interacts in a cooperative fashion with the locus, molding the functional behavior of the pore.     

Apical Ca2+-activated potassium channels in mouse parotid acinar cells

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.     

Large-conductance Ca2+-activated K+ channel beta1-subunit knockout mice are not hypertensive

Large-conductance Ca2+-activated K+ (BK) channels are composed of pore-forming α-subunits and accessory β1-subunits that modulate Ca2+ sensitivity. BK channels regulate arterial myogenic tone and renal Na+ clearance/K+ reabsorption. Previous studies using indirect or short-term blood pressure measurements found that BK channel β1-subunit knockout (BK β1-KO) mice were hypertensive. We evaluated 24-h mean arterial pressure (MAP) and heart rate in BK β1-KO mice using radiotelemetry. BK β1-KO mice did not have a higher 24-h average MAP when compared with wild-type (WT) mice, although MAP was ～10 mmHg higher at night. The dose-dependent peak declines in MAP by nifedipine were only slightly larger in BK β1-KO mice. In BK β1-KO mice, giving 1% NaCl to mice to drink for 7 days caused a transient (5 days) elevation of MAP (～5 mmHg); MAP returned to pre-saline levels by day 6. BK β1-KO mesenteric arteries in vitro demonstrated diminished contractile responses to paxilline, increased reactivity to Bay K 8644 and norepinephrine (NE), and maintained relaxation to isoproterenol. Paxilline and Bay K 8644 did not constrict WT or BK β1-KO mesenteric veins (MV). BK β1-subunits are not expressed in MV. The results indicate that BK β1-KO mice are not hypertensive on normal or high-salt intake. BK channel deficiency increases arterial reactivity to NE and L-type Ca2+ channel function in vitro, but the L-type Ca2+ channel modulation of MAP is not altered in BK β1-KO mice. BK and L-type Ca(2+) channels do not modulate murine venous tone. It appears that selective loss of BK channel function in arteries only is not sufficient to cause sustained hypertension.     

Bcl2 mitigates Ca2+ entry and mitochondrial Ca2+ overload through downregulation of L-type Ca2+ channels in PC12 cells

Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca(2+)](c)) are linked to neuronal apoptosis in epilepsy and in cerebral ischemia, respectively. Apoptotic programmed cell death is regulated by the antiapoptotic Bcl2 family of proteins. Here, we investigated the role of Bcl2 on calcium (Ca(2+)) homeostasis in PC12 cells, focusing on L-type voltage-dependent calcium channels (VDCC). Cytosolic Ca(2+) transients ([Ca(2+)](c)) and changes of mitochondrial Ca(2+) concentrations ([Ca(2+)](m)) were monitored using cytosolic and mitochondrially targeted aequorins of control PC12 cells and PC12 cells stably overexpressing Bcl2. We found that: (i) the [Ca(2+)](c) and [Ca(2+)](m) elevations elicited by K(+) pulses were markedly depressed in Bcl2 cells, with respect to control cells; (ii) such depression of [Ca(2+)](m) was not seen either in digitonin-permeabilized cells or in intact cells treated with ionomycin; (iii) the [Ca(2+)](c) transient depression seen in Bcl2 cells was reversed by shRNA transfection, as well as by the Bcl2 inhibitor HA14-1; (iv) the L-type Ca(2+) channel agonist Bay K 8644 enhanced K(+)-evoked [Ca(2+)](m) peak fourfold in Bcl2, and twofold in control cells; (v) in current-clamped cells the depolarization evoked by K(+) generated a more hyperpolarized voltage step in Bcl2, as compared to control cells. Taken together, our experiments suggest that the reduction of the [Ca(2+)](c) and [Ca(2+)](m) transients elicited by K(+), in PC12 cells overexpressing Bcl2, is related to the reduction of Ca(2+) entry through L-type Ca(2+) channels. This may be due to the fact that Bcl2 mitigates cell depolarization, thus diminishing the recruitment of L-type Ca(2+) channels, the subsequent Ca(2+) entry, and mitochondrial Ca(2+) overload.     

Reduced Ca2+-dependent activation of large-conductance Ca2+-activated K+ channels from arteries of Type 2 diabetic Zucker diabetic fatty rats

Although it is well established that diabetes impairs endothelium-dependent vasodilation, including those pathways involving vascular myocyte large-conductance Ca(2+)-activated K(+) channels (BK(Ca)), little is known about the effects of diabetes on BK(Ca) activation as an intrinsic response to contractile stimulation. We have investigated this mechanism in a model of Type 2 diabetes, the male Zucker diabetic fatty (ZDF) rat. BK(Ca) function in prediabetic (5-7 wk) and diabetic (17-20 wk) ZDF and lean control animals was assessed in whole arteries using myograph and electrophysiology techniques and in freshly dissociated myocytes by patch clamping. Log EC(25) values for phenylephrine concentration-tension curves were shifted significantly to the left by blockade of BK(Ca) with iberiotoxin (IBTX) in arteries from non- and prediabetic animals but not from diabetic animals. Smooth muscle hyperpolarizations of arteries evoked by the BK(Ca) opener NS-1619 were significantly reduced in the diabetic group. Voltage-clamp recordings indicated that IBTX-sensitive currents were not enhanced to the extent observed in nondiabetic controls by increasing the Ca(2+) concentration in the pipette solution or the application of NS-1619 in myocytes from diabetic animals. An alteration in the expression of BK(Ca) beta(1) subunits was not evident at either the mRNA or protein level in arteries from diabetic animals. Collectively, these results suggest that myocyte BK(Ca) of diabetic animals does not significantly oppose vasoconstriction, unlike that of prediabetic and control animals. This altered function was related to a reduced Ca(2+)-dependent activation of the channel not involving beta(1) subunits.