The potassium channel subunit Kvß1 serves as a major control point for synaptic facilitation.

Analysis of the presynaptic action potential's (APsyn) role in synaptic facilitation in hippocampal pyramidal neurons has been difficult due to size limitations of axons. We overcame these size barriers by combining high-resolution optical recordings of membrane potential, exocytosis, and Ca2+ in cultured hippocampal neurons. These recordings revealed a critical and selective role for Kv1 channel inactivation in synaptic facilitation of excitatory hippocampal neurons. Presynaptic Kv1 channel inactivation was mediated by the Kvß1 subunit and had a surprisingly rapid onset that was readily apparent even in brief physiological stimulation paradigms including paired-pulse stimulation. Genetic depletion of Kvß1 blocked all broadening of the APsyn during high-frequency stimulation and eliminated synaptic facilitation without altering the initial probability of vesicle release. Thus, using all quantitative optical measurements of presynaptic physiology, we reveal a critical role for presynaptic Kv channels in synaptic facilitation at presynaptic terminals of the hippocampus upstream of the exocytic machinery.

The ß4-Subunit of the Large-Conductance Potassium Ion Channel KCa1.1 Regulates Outflow Facility in Mice.

Purpose: The large-conductance calcium-activated potassium channel KCa1.1 (BKCa, maxi-K) influences aqueous humor outflow facility, but the contribution of auxiliary ß-subunits to KCa1.1 activity in the outflow pathway is unknown. Methods: Using quantitative polymerase chain reaction, we measured expression of ß-subunit genes in anterior segments of C57BL/6J mice (Kcnmb1-4) and in cultured human trabecular meshwork (TM) and Schlemm's canal (SC) cells (KCNMB1-4). We also measured expression of Kcnma1/KCNMA1 that encodes the pore-forming α-subunit. Using confocal immunofluorescence, we visualized the distribution of ß4 in the conventional outflow pathway of mice. Using iPerfusion, we measured outflow facility in enucleated mouse eyes in response to 100 or 500 nM iberiotoxin (IbTX; N = 9) or 100 nM martentoxin (MarTX; N = 12). MarTX selectively blocks ß4-containing KCa1.1 channels, whereas IbTX blocks KCa1.1 channels that lack ß4. Results: Kcnmb4 was the most highly expressed ß-subunit in mouse conventional outflow tissues, expressed at a level comparable to Kcnma1. ß4 was present within the juxtacanalicular TM, appearing to label cellular processes connecting to SC cells. Accordingly, KCNMB4 was the most highly expressed ß-subunit in human TM cells, and the sole ß-subunit in human SC cells. To dissect functional contribution, MarTX decreased outflow facility by 35% (27%, 42%; mean, 95% confidence interval) relative to vehicle-treated contralateral eyes, whereas IbTX reduced outflow facility by 16% (6%, 25%). Conclusions: The ß4-subunit regulates KCa1.1 activity in the conventional outflow pathway, significantly influencing outflow function. Targeting ß4-containing KCa1.1 channels may be a promising approach to lower intraocular pressure to treat glaucoma.

Estradiol-modified prolactin secretion independently of action potentials and Ca2+ and blockade of outward potassium currents in GH3 cells.

Estrogens facilitate prolactin (PRL) secretion acting on pituitary cells. In GH3 cells, estradiol induces acute action potentials and oscillations of intracellular Ca2+ associated with the secretagogue function. Estradiol modulates several ion channels which may affect the action potential rate and the release of PRL in lactotroph cells, which might depend on its concentration. The aims were to characterize the acute effect of supraphysiological concentrations of estradiol on Ca2+ and noninactivating K+ currents and measure the effect on the spontaneous action potentials and PRL release in the somatolactotroph cell line, GH3. Electrophysiological studies were carried out by voltage- and current-clamp techniques and ELISA determination of PRL secretion. Pharmacological concentrations of estradiol (above 1 µM), without a latency period, blocked Ca2+ channels and noninactivating K+ currents, including the large-conductance voltage- and Ca2+-activated K+ channels (BK), studied in whole-cell nystatin perforated and in excided inside-out patches of GH3 and CHO cells, transiently transfected with the human α-pore forming subunit of BK. The effect on BK was contrary to the agonist effect associated with the regulatory ß1-subunits of the BK, which GH3 cells lack, but its transient transfection did not modify the noninactivating current blockade, suggesting a different mechanism of regulation. Estradiol, at the same concentration range, acutely decreased the frequency of action potentials, an expected effect as consequence of the Ca2+ channel blockade. Despite this, PRL secretion initially increased, followed by a decrease in long-term incubations. This suggests that, in GH3 cells, supraphysiological concentrations of estradiol modulating PRL secretion are partially independent of extracellular Ca2+ influx.

Coronary arterial BK channel dysfunction exacerbates ischemia/reperfusion-induced myocardial injury in diabetic mice.

The large conductance Ca(2+)-activated K(+) (BK) channels, abundantly expressed in coronary artery smooth muscle cells (SMCs), play a pivotal role in regulating coronary circulation. A large body of evidence indicates that coronary arterial BK channel function is diminished in both type 1 and type 2 diabetes. However, the consequence of coronary BK channel dysfunction in diabetes is not clear. We hypothesized that impaired coronary BK channel function exacerbates myocardial ischemia/reperfusion (I/R) injury in streptozotocin-induced diabetic mice. Combining patch-clamp techniques and cellular biological approaches, we found that diabetes facilitated the colocalization of angiotensin II (Ang II) type 1 receptors and BK channel α-subunits (BK-α), but not BK channel ß1-subunits (BK-ß1), in the caveolae of coronary SMCs. This caveolar compartmentation in vascular SMCs not only enhanced Ang II-mediated inhibition of BK-α but also produced a physical disassociation between BK-α and BK-ß1, leading to increased infarct size in diabetic hearts. Most importantly, genetic ablation of caveolae integrity or pharmacological activation of coronary BK channels protected the cardiac function of diabetic mice from experimental I/R injury in both in vivo and ex vivo preparations. Our results demonstrate a vascular ionic mechanism underlying the poor outcome of myocardial injury in diabetes. Hence, activation of coronary BK channels may serve as a therapeutic target for cardiovascular complications of diabetes.

BK channel ß1-subunit deficiency exacerbates vascular fibrosis and remodelling but does not promote hypertension in high-fat fed obesity in mice.

OBJECTIVE: Reduced expression or increased degradation of BK (large conductance Ca-activated K) channel ß1-subunits has been associated with increased vascular tone and hypertension in some metabolic diseases. The contribution of BK channel function to control of blood pressure (BP), heart rate (HR) and vascular function/structure was determined in wild-type and BK channel ß1-subunit knockout mice fed a high-fat or control diet. METHODS AND RESULTS: After 24 weeks of high-fat diet, wild-type and BK ß1-knockout mice were obese, diabetic, but normotensive. High-fat-BK ß1-knockout mice had decreased HR, while high-fat-wild-type mice had increased HR compared with mice on the control diet. Ganglion blockade caused a greater fall in BP and HR in mice on a high-fat diet than in mice on the control diet. ß1-adrenergic receptor blockade reduced BP and HR equally in all groups. α1-adrenergic receptor blockade decreased BP in high-fat-BK ß1-knockout mice only. Echocardiographic evaluation revealed left ventricular hypertrophy in high-fat-BK ß1-knockout mice. Although under anaesthesia, mice on a high-fat diet had higher absolute stroke volume and cardiac output, these measures were similar to control mice when adjusted for body weight. Mesenteric arteries from high-fat-BK ß1-knockout mice had higher norepinephrine reactivity, greater wall thickness and collagen accumulation than high-fat-wild-type mesenteric arteries. Compared with control-wild-type mesenteric arteries, high-fat-wild-type mesenteric arteries had blunted contractile responses to a BK channel blocker, although BK α-subunit (protein) and ß1-subunit (mRNA) expression were unchanged. CONCLUSION: BK channel deficiency promotes increased sympathetic control of BP, and vascular dysfunction, remodelling and fibrosis, but does not cause hypertension in high-fat fed mice.

Recombinant expression and functional characterization of martentoxin: a selective inhibitor for BK channel (α + ß4).

Martentoxin (MarTX), a 37-residue peptide purified from the venom of East-Asian scorpion (Buthus martensi Karsch), was capable of blocking large-conductance Ca2+-activated K+ (BK) channels. Here, we report an effective expression and purification approach for this toxin. The cDNA encoding martentoxin was expressed by the prokaryotic expression system pGEX-4T-3 which was added an enterokinase cleavage site by PCR. The fusion protein (GST-rMarTX) was digested by enterokinase to release hetero-expressed toxin and further purified via reverse-phase HPLC. The molecular weight of the hetero-expressed rMarTX was 4059.06 Da, which is identical to that of the natural peptide isolated from scorpion venom. Functional characterization through whole-cell patch clamp showed that rMarTX selectively and potently inhibited the currents of neuronal BK channels (α + ß4) (IC50 = 186 nM), partly inhibited mKv1.3, but hardly having any significant effect on hKv4.2 and hKv3.1a even at 10 µM. Successful expression of martentoxin lays basis for further studies of structure-function relationship underlying martentoxin or other potassium-channel specific blockers.

Distinct activity of BK channel ß1-subunit in cerebral and pulmonary artery smooth muscle cells.

This study was designed to test a hypothesis that the functional activity of big-conductance, Ca(2+)-activated K(+) (BK) channels is different in cerebral and pulmonary artery smooth muscle cells (CASMCs and PASMCs). Using patch-clamp recordings, we found that the activity of whole cell and single BK channels were significantly higher in CASMCs than in PASMCs. The voltage and Ca(2+) sensitivity of BK channels were greater in CASMCs than in PASMCs. Targeted gene knockout of ß(1)-subunits significantly reduced BK currents in CASMCs but had no effect in PASMCs. Western blotting experiments revealed that BK channel α-subunit protein expression level was comparable in CASMCs and PASMCs; however, ß(1)-subunit protein expression level was higher in CASMCs than in PASMCs. Inhibition of BK channels by the specific blocker iberiotoxin enhanced norepinephrine-induced increase in intracellular calcium concentration in CASMCs but not in PASMCs. Systemic artery blood pressure was elevated in ß(1)(-/-) mice. In contrast, pulmonary artery blood pressure was normal in ß(1)(-/-) mice. These findings provide the first evidence that the activity of BK channels is higher in cerebral than in PASMCs. This heterogeneity is primarily determined by the differential ß(1)-subunit function and contributes to diverse cellular responses in these two distinct types of cells.

Chronic hypoxia suppresses pregnancy-induced upregulation of large-conductance Ca2+-activated K+ channel activity in uterine arteries.

Our previous study demonstrated that increased Ca(2+)-activated K(+) (BK(Ca)) channel activity played a key role in the normal adaptation of reduced myogenic tone of uterine arteries in pregnancy. The present study tested the hypothesis that chronic hypoxia during gestation inhibits pregnancy-induced upregulation of BK(Ca) channel function in uterine arteries. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep maintained at sea level (≈ 300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. Hypoxia during gestation significantly inhibited pregnancy-induced upregulation of BK(Ca) channel activity and suppressed BK(Ca) channel current density in pregnant uterine arteries. This was mediated by a selective downregulation of BK(Ca) channel ß1 subunit in the uterine arteries. In accordance, hypoxia abrogated the role of the BK(Ca) channel in regulating pressure-induced myogenic tone of uterine arteries that was significantly elevated in pregnant animals acclimatized to chronic hypoxia. In addition, hypoxia abolished the steroid hormone-mediated increase in the ß1 subunit and BK(Ca) channel current density observed in nonpregnant uterine arteries. Although the activation of protein kinase C inhibited BK(Ca) channel current density in pregnant uterine arteries of normoxic sheep, this effect was ablated in the hypoxic animals. The results demonstrate that selectively targeting BK(Ca) channel ß1 subunit plays a critical role in the maladaption of uteroplacental circulation caused by chronic hypoxia, which contributes to the increased incidence of preeclampsia and fetal intrauterine growth restriction associated with gestational hypoxia.

Requirement for functional BK channels in maintaining oscillation in venomotor tone revealed by species differences in expression of the ß1 accessory subunits.

We determined the possible role of large-conductance Ca2+-activated K (BK) channels in regulation of venous tone in small capacitance veins and blood pressure. In rat mesenteric venous smooth muscle cells (MV SMC), BK channel α- and ß1-subunits were coexpressed, unitary BK currents were detected, and single-channel currents were sensitive to voltage and [Ca2+]i. Rat MV SMCs displayed Ca sparks and iberiotoxin-sensitive spontaneous transient outward currents. Under resting conditions in vitro, rat MV exhibited nifedipine-sensitive spontaneous oscillatory constrictions. Blockade of BK channels by paxilline and Ca2+ sparks by ryanodine constricted rat MV. Nifedipine caused venodilation and blocked paxilline-induced, KCl-induced (20 mM), and BayK8644-induced contraction. Acute inhibition of BK channels with iberiotoxin in vivo increased blood pressure and reduced venous capacitance, measured as an increase in mean circulatory filling pressure in conscious rats. BK channel α-subunits and L-type Ca2+ channel α1-C subunits are expressed in murine MV. However, these channels are not functional because murine MV lack nifedipine-sensitive basal tone and rhythmic constrictions. Murine MV were also insensitive to paxilline, ryanodine, KCl, and BayK8644, consistent with our previous studies showing that murine MV do not have BK ß1-subunits. These data show that not only there are species-dependent properties in ion channel control of venomotor tone but also BK channels are required for rhythmic oscillations in venous tone.

High glucose alters apoptosis and proliferation in HEK293 cells by inhibition of cloned BK Ca channel.

It has been reported that diabetic vascular dysfunction is associated with impaired function of large conductance Ca(2+) -activated K(+) (BK(Ca) ) channels. However, it is unclear whether impaired BK(Ca) channel directly participates in regulating diabetic vascular remodeling by altering cell growth in response to hyperglycemia. In the present study, we investigated the specific role of BK(Ca) channel in controlling apoptosis and proliferation under high glucose concentration (25 mM). The cDNA encoding the α+ß1 subunit of BK(Ca) channel, hSloα+ß1, was transiently transfected into human embryonic kidney 293 (HEK293) cells. Cloned BK(Ca) currents were recorded by both whole-cell and cell-attached patch clamp techniques. Cell apoptosis was assessed with immunocytochemistry and analysis of fragmented DNA by agarose gel electrophoresis. Cell proliferation was investigated by flow cytometry assays, MTT test, and immunocytochemistry. In addition, the expression of anti-apoptotic protein Bcl-2, intracellular Ca(2+) , and mitochondrial membrane potential (Δψm) were also examined to investigate the possible mechanisms. Our results indicate that inhibition of cloned BK(Ca) channels might be responsible for hyperglycemia-altered apoptosis and proliferation in HEK-hSloα+ß1 cells. However, activation of BK(Ca) channel by NS1619 or Tamoxifen significantly induced apoptosis and suppressed proliferation in HEK-hSloα+ß1 cells under hyperglycemia condition. When rat cerebral smooth muscle cells were cultured in hyperglycemia, similar findings were observed. Moreover, the possible mechanisms underlying the activation of BK(Ca) channel were associated with decreased expression of Bcl-2, elevation of intracellular Ca(2+) , and a concomitant depolarization of Δψm in HEK-hSloα+ß1 cells. In conclusion, cloned BK(Ca) channel directly regulated apoptosis and proliferation of HEK293 cell under hyperglycemia condition.

BK channel ß1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization.

The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific ß1 subunit regulate excitation­contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel ß1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK ß1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of ß1 knockout is eliminated by specific M2 receptor antagonism. The role of BK ß1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with ß1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK ß1 knockout or by paxilline block of BK channels. Normalization of ß1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/ß1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation­contraction coupling to more negative voltage ranges.

Tamoxifen inhibits BK channels in chick cochlea without alterations in voltage-dependent activation.

Large-conductance, Ca(2+)-activated, and voltage-gated potassium channels (BK, BK(Ca), or Maxi-K) play an important role in electrical tuning in nonmammalian vertebrate hair cells. Systematic changes in tuning frequency along the tonotopic axis largely result from variations in BK channel kinetics, but the molecular changes underpinning these functional variations remain unknown. Auxiliary beta(1) have been implicated in low-frequency tuning at the cochlear apex because these subunits dramatically slow channel kinetics. Tamoxifen (Tx), a (xeno)estrogen compound known to activate BK channels through the beta-subunit, was used to test for the functional presence of beta(1). The hypotheses were that Tx would activate the majority of BK channels in hair cells from the cochlear apex due to the presence of beta(1) and that the level of activation would exhibit a tonotopic gradient following the expression profile of beta(1). Outside-out patches of BK channels were excised from tall hair cells along the apical half of the chicken basilar papilla. In low-density patches, single-channel conductance was reduced and the averaged open probability was unaffected by Tx. In high-density patches, the amplitude of ensemble-averaged BK current was inhibited, whereas half-activation potential and activation kinetics were unaffected by Tx. In both cases, no tonotopic Tx-dependent activation of channel activity was observed. Therefore, contrary to the hypotheses, electrophysiological assessment suggests that molecular mechanisms other than auxiliary beta-subunits are involved in generating a tonotopic distribution of BK channel kinetics and electric tuning in chick basilar papilla.

Compartmentalized beta subunit distribution determines characteristics and ethanol sensitivity of somatic, dendritic, and terminal large-conductance calcium-activated potassium channels in the rat central nervous system.

Neurons are highly differentiated and polarized cells, whose various functions depend upon the compartmentalization of ion channels. The rat hypothalamic-neurohypophysial system (HNS), in which cell bodies and dendrites reside in the hypothalamus, physically separated from their nerve terminals in the neurohypophysis, provides a particularly powerful preparation in which to study the distribution and regional properties of ion channel proteins. Using electrophysiological and immunohistochemical techniques, we characterized the large-conductance calcium-activated potassium (BK) channel in each of the three primary compartments (soma, dendrite, and terminal) of HNS neurons. We found that dendritic BK channels, in common with somatic channels but in contrast to nerve terminal channels, are insensitive to iberiotoxin. Furthermore, analysis of dendritic BK channel gating kinetics indicates that they, like somatic channels, have fast activation kinetics, in contrast to the slow gating of terminal channels. Dendritic and somatic channels are also more sensitive to calcium and have a greater conductance than terminal channels. Finally, although terminal BK channels are highly potentiated by ethanol, somatic and dendritic channels are insensitive to the drug. The biophysical and pharmacological properties of somatic and dendritic versus nerve terminal channels are consistent with the characteristics of exogenously expressed alphabeta1 versus alphabeta4 channels, respectively. Therefore, one possible explanation for our findings is a selective distribution of auxiliary beta1 subunits to the somatic and dendritic compartments and beta4 to the terminal compartment. This hypothesis is supported immunohistochemically by the appearance of distinct punctate beta1 or beta4 channel clusters in the membrane of somatic and dendritic or nerve terminal compartments, respectively.

N-terminal inactivation domains of beta subunits are protected from trypsin digestion by binding within the antechamber of BK channels.

N termini of auxiliary beta subunits that produce inactivation of large-conductance Ca(2+)-activated K(+) (BK) channels reach their pore-blocking position by first passing through side portals into an antechamber separating the BK pore module and the large C-terminal cytosolic domain. Previous work indicated that the beta2 subunit inactivation domain is protected from digestion by trypsin when bound in the inactivated conformation. Other results suggest that, even when channels are closed, an inactivation domain can also be protected from digestion by trypsin when bound within the antechamber. Here, we provide additional tests of this model and examine its applicability to other beta subunit N termini. First, we show that specific mutations in the beta2 inactivation segment can speed up digestion by trypsin under closed-channel conditions, supporting the idea that the beta2 N terminus is protected by binding within the antechamber. Second, we show that cytosolic channel blockers distinguish between protection mediated by inactivation and protection under closed-channel conditions, implicating two distinct sites of protection. Together, these results confirm the idea that beta2 N termini can occupy the BK channel antechamber by interaction at some site distinct from the BK central cavity. In contrast, the beta 3a N terminus is digested over 10-fold more quickly than the beta2 N terminus. Analysis of factors that contribute to differences in digestion rates suggests that binding of an N terminus within the antechamber constrains the trypsin accessibility of digestible basic residues, even when such residues are positioned outside the antechamber. Our analysis indicates that up to two N termini may simultaneously be protected from digestion. These results indicate that inactivation domains have sites of binding in addition to those directly involved in inactivation.

The molecular mechanism of "ryegrass staggers," a neurological disorder of K+ channels.

"Ryegrass staggers" is a neurological condition of unknown mechanism that impairs motor function in livestock. It is caused by infection of perennial ryegrass pastures by an endophytic fungus that produces neurotoxins, predominantly the indole-diterpenoid compound lolitrem B. Animals grazing on such pastures develop uncontrollable tremors and become uncoordinated in their movement. Lolitrem B and the structurally related tremor inducer paxilline both act as potent large conductance calcium-activated potassium (BK) channel inhibitors. Using patch clamping, we show that their different apparent affinities correlate with their toxicity in vivo. To investigate whether the motor function deficits produced by lolitrem B and paxilline are due to inhibition of BK ion channels, their ability to induce tremor and ataxia in mice deficient in this ion channel (Kcnma1(-/-)) was examined. Our results show that mice lacking Kcnma1 are unaffected by these neurotoxins. Furthermore, doses of these substances known to be lethal to wild-type mice had no effect on Kcnma1(-/-) mice. These studies reveal the BK channel as the molecular target for the major components of the motor impairments induced by ryegrass neurotoxins. Unexpectedly, when the response to lolitrem B was examined in mice lacking the beta4 BK channel accessory subunit (Kcnmb4(-/-)), only low-level ataxia was observed. Our study therefore reveals a new role for the accessory BK beta4 subunit in motor control. The beta4 subunit could be considered as a potential target for treatment of ataxic conditions in animals and in humans.

Structural basis for toxin resistance of beta4-associated calcium-activated potassium (BK) channels.

The functional diversity of large conductance Ca(2+)- and voltage-dependent K(+) (BK) channels arises mainly from co-assembly of the pore-forming mSlo alpha subunits with four tissue-enriched auxiliary beta subunits. The structural basis of the interaction between alpha subunits with beta subunits is not well understood. Using computational and experimental methods, we demonstrated that four mSlo turrets decentralized distally from the channel pore to provide a wide open conformation and that the mSlo and hbeta4 subunits together formed a "helmet" containing three basic residues (Lys-120, Arg-121, and Lys-125), which impeded the entry of charybdotoxin (ChTX) by both the electrostatic interaction and limited space. In addition, the tyrosine insert mutant (in100Y) showed 56% inhibition, with a K(d) = 17 nm, suggesting that the hbeta4 lacks an external ChTX-binding site (Tyr-100). We also found that mSlo had an internal binding site (Tyr-294) in the alpha subunits that could "permanently" block 15% of mSlo+hbeta4 currents in the presence of 100 nm ChTX. These findings provide a better understanding of the diverse interactions between alpha and beta subunits and will improve the design of channel inhibitors.

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).

Diabetes downregulates large-conductance Ca2+-activated potassium beta 1 channel subunit in retinal arteriolar smooth muscle.

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca(2+) release events (Ca(2+)-sparks), trigger the activation of large-conductance Ca(2+)-activated K(+)(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca(2+)](i) release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca(2+)-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BKalpha subunit was unchanged. The Ca(2+)-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BKbeta1 subunit confers Ca(2+)-sensitivity to BK channel complexes and both transcript and protein levels for BKbeta1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BKbeta1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.