Distinct regions of the slo subunit determine differential BKCa channel responses to ethanol

BACKGROUND: Ethanol at clinically relevant concentrations increases BKCa channel activity in dorsal root ganglia neurons, GH3 cells, and neurohypophysial terminals, leading to decreases in cell excitability and peptide release. In contrast, ethanol inhibits BKCa channels from aortic myocytes, which likely contributes to alcohol-induced aortic constriction. The mechanisms that determine differential BKCa channel responses to ethanol are unknown. We hypothesized that nonconserved regions in the BKCa channel-forming subunit (slo) are major contributors to the differential alcohol responses of different BKCa channel phenotypes. METHODS: We constructed chimeras by interchanging the core and the tail domains of two BKCa channel-forming subunits (mslo and bslo) that, after expression, differentially respond to ethanol (activation and inhibition, respectively), and studied ethanol action on these mbslo and bmslo chimeric channels using single-channel, patch-clamp techniques. RESULTS AND CONCLUSION: Data from cell-free membranes patches demonstrate that the activity of channels that share a mslo-type core-linker (wt mslo and the mbslo chimera) is consistently and significantly potentiated by acute exposure to ethanol. Thus, a mslo tail is not necessary for ethanol potentiation of slo channels. In contrast, the activity of channels that share a bslo-type core-linker (wt bslo and the bmslo chimera) display heterogenous responses to ethanol: inhibition (in the majority of cases), refractoriness, or activation. Overall, our data indicate that the slo core-linker is a critical region likely contributing to the differential responses of BKCa channels to ethanol.     

Sizing up Ethanol-Induced Plasticity: The Role of Small and Large Conductance Calcium-Activated Potassium Channels

Small (SK) and large conductance (BK) Ca²⁺-activated K⁺ channels contribute to action potential repolarization, shape dendritic Ca²⁺spikes and postsynaptic responses, modulate the release of hormones and neurotransmitters, and contribute to hippocampal-dependent synaptic plasticity. Over the last decade, SK and BK channels have emerged as important targets for the development of acute ethanol tolerance and for altering neuronal excitability following chronic ethanol consumption. In this mini-review, we discuss new evidence implicating SK and BK channels in ethanol tolerance and ethanol-associated homeostatic plasticity. Findings from recent reports demonstrate that chronic ethanol produces a reduction in the function of SK channels in VTA dopaminergic and CA1 pyramidal neurons. It is hypothesized that the reduction in SK channel function increases the propensity for burst firing in VTA neurons and increases the likelihood for aberrant hyperexcitability during ethanol withdrawal in hippocampus. There is also increasing evidence supporting the idea that ethanol sensitivity of native BK channel results from differences in BK subunit composition, the proteolipid microenvironment, and molecular determinants of the channel-forming subunit itself. Moreover, these molecular entities play a substantial role in controlling the temporal component of ethanol-associated neuroadaptations in BK channels. Taken together, these studies suggest that SK and BK channels contribute to ethanol tolerance and adaptive plasticity.     

The slowpoke gene is necessary for rapid ethanol tolerance in Drosophila

BACKGROUND: Ethanol is one of the most commonly used drugs in the world. We are interested in the compensatory mechanisms used by the nervous system to counter the effects of ethanol intoxication. Recently, the slowpoke BK-type calcium-activated potassium channel gene has been shown to be involved in ethanol sensitivity in Caenorhabditis elegans and in rapid tolerance to the anesthetic benzyl alcohol in Drosophila. METHODS: We used Drosophila mutants to investigate the role of slowpoke in rapid tolerance to sedation with ethanol vapor. Rapid tolerance was defined as a reduction in the sedative phase caused by a single previous sedation. The ethanol and water contents of flies were measured to determine if pharmacodynamic changes could account for tolerance. RESULTS: A saturated ethanol air stream caused sedation in <20 min and resulted in rapid tolerance that was apparent 4 hr after sedation. Two independently isolated null mutations in the slowpoke gene eliminated the capacity for tolerance. In addition, a third mutation that blocked expression specifically in the nervous system also blocked rapid tolerance. Water measurements showed that both ethanol and mock sedation caused equivalent dehydration. Furthermore, a single prior exposure to ethanol did not cause a change in the ethanol clearance rate. CONCLUSIONS: Rapid tolerance, measured as a reduction in the duration of sedation, is a pharmacokinetic response to ethanol that does not occur without slowpoke expression in the nervous system in Drosophila. The slowpoke channel must be involved in triggering or producing a homeostatic mechanism that opposes the sedative effects of ethanol.     

Essential role for smooth muscle BK channels in alcohol-induced cerebrovascular constriction

Binge drinking is associated with increased risk for cerebrovascular spasm and stroke. Acute exposure to ethanol at concentrations obtained during binge drinking constricts cerebral arteries in several species, including humans, but the mechanisms underlying this action are largely unknown. In a rodent model, we used fluorescence microscopy, patch-clamp electrophysiology, and pharmacological studies in intact cerebral arteries to pinpoint the molecular effectors of ethanol cerebrovascular constriction. Clinically relevant concentrations of ethanol elevated wall intracellular Ca(2+) concentration and caused a reversible constriction of cerebral arteries (EC(50) = 27 mM; E(max) = 100 mM) that depended on voltage-gated Ca(2+) entry into myocytes. However, ethanol did not directly increase voltage-dependent Ca(2+) currents in isolated myocytes. Constriction occurred because of an ethanol reduction in the frequency (-53%) and amplitude (-32%) of transient Ca(2+)-activated K(+) (BK) currents. Ethanol inhibition of BK transients was caused by a reduction in Ca(2+) spark frequency (-49%), a subsarcolemmal Ca(2+) signal that evokes the BK transients, and a direct inhibition of BK channel steady-state activity (-44%). In contrast, ethanol failed to modify Ca(2+) waves, a major vasoconstrictor mechanism. Selective block of BK channels largely prevented ethanol constriction in pressurized arteries. This study pinpoints the Ca(2+) spark/BK channel negative-feedback mechanism as the primary effector of ethanol vasoconstriction.     

Beta-subunits are important modulators of the acute response to alcohol in human BK channels

BACKGROUND: The BK channel (a Ca2+-activated potassium ion channel encoded by the slo gene) has been defined as a target of alcohol action in a number of preparations, possibly serving as primary mediator of intoxication in the Caenorhabditis elegans model system. However, we know little of the actions of alcohol on human BK, nor the consequences of BK subunit composition on alcohol action. METHODS: Here, we use human embryonic kidney (HEK) cells to express various subunit combinations (hslo alpha+beta1 or beta4) of human BK, and examine the acute actions of alcohol on this channel using single channel recording techniques. RESULTS: The human channel is potentiated by alcohol, although the presence of the beta1, and to a lesser extent, beta4-subunit, significantly reduced acute ethanol potentiation. Potentiation increased with concentration up to an asymptote, at which point potentiation decreased. The concentration of the asymptote differed according to subunit composition. The mechanism of potentiation was also subunit-dependent, with 25 mM ethanol affecting the mean open time of hSlo+beta4 channels, whereas channel open time was unaffected by the presence of beta1. The possibility that the known effect of the beta-subunit on calcium sensitivity accounts for its modulation of acute alcohol action is discussed. CONCLUSION: Our data reinforce the idea that, as in other systems, BK may play a major role in alcohol's actions in humans, and highlight the potential role of channel subunit composition in the response to alcohol.     

An alcohol-sensing site in the calcium- and voltage-gated,large conductance potassium (BK) channel

Ethanol alters BK (slo1) channel function leading to perturbation of physiology and behavior. Site(s) and mechanism(s) of ethanol–BK channel interaction are unknown. We demonstrate that ethanol docks onto a water-accessible site that is strategically positioned between the slo1 calcium-sensors and gate. Ethanol only accesses this site in presence of calcium, the BK channel’s physiological agonist. Within the site, ethanol hydrogen-bonds with K361. Moreover, substitutions that hamper hydrogen bond formation or prevent ethanol from accessing K361 abolish alcohol action without altering basal channel function. Alcohol interacting site dimensions are approximately 10.7 × 8.6 × 7.1 Å, accommodating effective (ethanol-heptanol) but not ineffective (octanol, nonanol) channel activators. This study presents: (i) to our knowledge, the first identification and characterization of an n-alkanol recognition site in a member of the voltage-gated TM6 channel superfamily; (ii) structural insights on ethanol allosteric interactions with ligand-gated ion channels; and (iii) a first step for designing agents that antagonize BK channel-mediated alcohol actions without perturbing basal channel function.Alcohol (ethyl alcohol, ethanol) is a psychoactive agent that has been overwhelmingly consumed by mankind across cultures and civilizations. Alcohol actions on central nervous system (CNS) physiology and behavior are largely independent of beverage type but due to ethanol itself (1). Ethanol alters cell excitability by modifying function of transmembrane (TM) ion channel proteins, including K⁺ channels. These channels constitute the most heterogeneous and extensive group of ion channels, its members belonging to TM2, TM4, and TM6 protein superfamilies. Within this myriad of proteins, several K⁺ channels have been shown to modify behavior in response to acute exposure to ethanol concentrations that reach the CNS and other excitable tissues during alcohol drinking (2–5). However, with the sole exception of the TM2, G protein-regulated inward rectifier K⁺ (GIRK) channel (6), there is no structural information on ethanol-K⁺ channel protein interacting sites currently available.Voltage/Ca²⁺-gated, large conductance K⁺ channels (BK), which are members of the TM6 voltage-gated ion channel superfamily, constitute major mediators of alcohol actions in excitable tissues. Acute exposure to ethanol levels reached in CNS during alcohol intoxication alters BK-mediated currents and thus, elicits widespread and profound modifications in physiology and behavior. In rodent models, acute ethanol exposure leads to reduced vasopressin, oxytocin and growth hormone release with consequent perturbation in physiology and behavior (7), altered firing rates in nucleus accumbens (8) and dorsal root ganglia neurons (9), and alcohol-induced cerebral artery constriction (10, 11). Moreover, studies in both mammals and invertebrate models demonstrate that ethanol targeting of neuronal BK is involved in development of alcohol tolerance and dependence (12–16). Although the physiological and behavioral consequences of ethanol disruption of BK function have been well documented, it remains unknown whether alcohol modification of BK function results from drug interaction with a defined recognition site(s) in a protein target vs. physical perturbation of the proteolipid environment where the BK protein resides. Thus, location and structural characteristics of the ethanol-recognition site(s), as well as nature of chemical bonds between ethanol and functional target that lead to modification of BK function, remain unknown.Ethanol-induced regulation of BK channels is fine-tuned by many factors, including the BK channel-forming slo1 protein (α subunit) isoform (17) and its modification by phosphorylation (18), BK channel accessory (β) subunits (11), the channel-activating ionic ligand (Cai2+) (19) and the lipid microenvironment around the BK protein complex (20). However, ethanol perturbation of BK function is sustained when the slo1 protein is probed with the alcohol in cell-free membrane patches (19–21) or after protein reconstitution into artificial lipid bilayers (22). We recently demonstrated that perturbation of slo1 function by ethanol concentrations reached in blood during alcohol intoxication does not extend to Na⁺-gated slo2 and pH-gated slo3 channels, which are phylogenetically and structurally related to slo1. However, ethanol sensitivity does extend to a prokaryotic K⁺ channel from Methanobacterium thermoautotrophicum (MthK) (23), a TM2 ion channel that shares basic Cai2+-driven gating mechanisms with slo1 (24). Collectively, these studies lead us to hypothesize that ethanol-recognition site(s) involved in alcohol modification of BK current exists in the slo1 cytosolic Cai2+-sensing tail domain (CTD).Based on crystallographic data of the slo1 CTD and primary alignment of slo1-related ion channels that share ethanol sensitivity, we first identified eight putative ethanol recognition regions in the slo1 CTD. Using computational modeling, point amino acid substitutions and electrophysiology, we identified a distinct pocket as the ethanol-recognition site that leads to alcohol modification of BK current. This site has a few common characteristics of alcohol-binding protein sequences (25), yet presents features that differ from those of the alcohol site described in GIRK (6). In opposition to GIRK currents, which can be potentiated by alcohol in absence of G proteins (6), ethanol modulation of BK currents is dependent on the presence of Cai2+ (19). Our data strongly suggest that ethanol access to the newly identified BK ethanol-recognition site depends on the Cai2+ levels associated with the slo1 CTD. Thus, current data not only provide a structural basis for understanding Cai2+–alcohol allosterism on BK channels but could render structural insights on other ligand-gated channels that are activated by ethanol in presence of their natural ligand (26–30). Finally, present data document that the newly identified site plays a critical role in BK channel sensitivity to long-chain alkanols and explain the reported chain length differential sensitivity (“cutoff”) of linear n-alkanols to modify BK current (31).Identification of a distinct alcohol-sensing site in BK channels opens the door for rational design of pharmaceuticals to counteract widespread effects of alcohol intoxication in the body without altering basal BK channel function. Because this site is present in human BK protein ({"type":"entrez-protein","attrs":{"text":"AAA92290.1","term_id":"758791","term_text":"AAA92290.1"}}AAA92290.1), it is possible that genetic, epigenetic or other modifications of the alcohol-sensing site in BK channels could contribute to differential sensitivity to alcohol intoxication in humans. In addition, considering that individuals with low alcohol sensitivity are prone to developing heavy drinking (32), an altered profile of alcohol-sensing site on BK channels might be included as a potential predictor, along with other targets, for developing alcohol preference.     

Carbon monoxide stimulates the Ca2(+)-activated big conductance k channels in cultured human endothelial cells

We used the whole-cell patch-clamp technique to study K channels in the human umbilical vein endothelial cells and identified a 201 pS K channel, which was blocked by tetraethylammonium and iberiotoxin but not by TRAM34 and apamin. This suggests that the Ca(2+)-activated big-conductance K channel (BK) is expressed in endothelial cells. Application of carbon monoxide (CO) or tricarbonylchloro(glycinato)ruthenium(II), a water soluble CO donor, stimulated the BK channels. Moreover, application of hemin, a substrate of heme oxygenase, mimicked the effect of CO and increased the BK channel activity. The stimulatory effect of hemin was significantly diminished by tin mesoporphyrin, an inhibitor of heme oxygenase. To determine whether the stimulatory effect of CO on the BK channel was mediated by NO and the cGMP-dependent pathway, we examined the effect of CO on BK channels in cells treated with, N(G)-nitro-l-arginine methyl ester, 1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, or KT5823, an inhibitor of protein kinase G. Addition of either diethylamine NONOate or sodium nitroprusside significantly increased BK channel activity. Inhibition of endogenous NO synthesis with N(G)-nitro-l-arginine methyl ester, blocking soluble guanylate cyclase or protein kinase G, delayed but did not prevent the CO-induced activation of BK channels. Finally, application of an antioxidant agent, ebselen, had no effect on CO-mediated stimulation of BK channels in human umbilical vein endothelial cells. We conclude that BK channels are expressed in human umbilical vein endothelial cells and that they are activated by both CO and NO. CO activates BK channels directly, as well as via a mechanism involving NO or the cGMP-dependent pathway.     

SK4通道和BK通道对心脏起搏功能的影响

钙离子激活的钾离子通道根据其电导的大小主要分为三种类型：小电导钙离子激活的钾离子通道SK（SK1-3）,中电导钙离子激活的钾离子通道(IK或SK4)和大电导钙离子激活的钾离子通道(BK)。既往研究已表明SK、SK4和BK在心律失常,高血压,心肌再灌注损伤等的发生发展中发挥重要作用。最近研究提示SK4和BK具有调节心脏起搏的功能。本文旨在综述这两种离子通道在心脏起搏中的作用和机制及构建生物起搏的潜在可能     

Melatonin ameliorates hippocampal nitric oxide production and large conductance calcium-activated potassium channel activity in chronic intermittent hypoxia

Melatonin protects against hippocampal injury induced by intermittent hypoxia (IH). IH-induced oxidative stress is associated with decreases in constitutive production of nitric oxide (NO) and in the activity of large conductance calcium-activated potassium (BK) channels in hippocampal neurons. We tested the hypothesis that administration of melatonin alleviates the NO deficit and impaired BK channel activity in the hippocampus of IH rats. Sprague-Dawley rats were injected with melatonin (10 mg/kg, i.p.) or vehicle before daily IH exposure for 8 hr for 7 days. The NO and intracellular calcium ([Ca2+]i) levels in the CA1 region of hippocampal slices were measured by electrochemical microsenor and spectrofluorometry, respectively. The activity of BK channels was recorded by patch-clamping electrophysiology in dissociated CA1 neurons. Malondialdehyde levels were increased in the hippocampus of hypoxic rats and were lowered by the melatonin treatment. Levels of NO under resting and hypoxic conditions, and the protein expression of neuronal NO synthase (nNOS) were significantly reduced in the CA1 neurons of hypoxic animals compared with the normoxic controls. These deficits were mitigated in the melatonin-treated hypoxic rats with an improved [Ca2+]i response to acute hypoxia. The open probability of BK channels was decreased in the hypoxic rats and was partially restored in the melatonin-treated animals, without alterations in the expression of channel subunits and unitary conductance. Acute treatment of melatonin had no significant effects on the BK channel activity or on the [Ca2+]i response to hypoxia. Collectively, these results suggest that melatonin ameliorates the constitutive NO production and BK channel activity via an antioxidant mechanism against an IH-induced down-regulation of nNOS expression in hippocampal neurons.     

Identification of a BK channel auxiliary protein controlling molecular and behavioral tolerance to alcohol

Tolerance, described as the loss of drug effectiveness over time, is an important component of addiction. The degree of acute behavioral tolerance to alcohol exhibited by a naïve subject can predict the likelihood of alcohol abuse. Thus, the determinants of acute tolerance are important to understand. Calcium- and voltage-gated (BK) potassium channels, consisting of pore forming α and modulatory β subunits, are targets of ethanol (EtOH) action. Here, we examine the role, at the molecular, cellular, and behavioral levels, of the BK β4 subunit in acute tolerance. Single channel recordings in HEK-293 cells show that, in the absence of β4, EtOH potentiation of activity exhibits acute tolerance, which is blocked by coexpressing the β4 subunit. BK channels in acutely isolated medium spiny neurons from WT mice (in which the β4 subunit is well-represented) exhibit little tolerance. In contrast, neuronal BK channels from β4 knockout (KO) mice do display acute tolerance. Brain slice recordings showed tolerance to EtOH's effects on spike patterning in KO but not in WT mice. In addition, β4 KO mice develop rapid tolerance to EtOH's locomotor effects, whereas WT mice do not. Finally, in a restricted access ethanol self-administration assay, β4 KO mice drink more than their WT counterparts. Taken together, these data indicate that the β4 subunit controls ethanol tolerance at the molecular, cellular, and behavioral levels, and could determine individual differences in alcohol abuse and alcoholism, as well as represent a therapeutic target for alcoholism.     

Dopamine transporters depolarize neurons by a channel mechanism

Neurotransmitter transporters generate larger currents than expected if one assumes fixed stoichiometry models. It remains controversial, however, whether these depolarizing currents arise from high density and rapid turnover rates of a classical transporter, or whether transporters exhibit bona fide channel behavior. Although heterologously expressed transporters show single-channel behavior and noise analysis in native cells strongly suggests channel behavior, no directly observed single-channel events associated with transporters have been reported thus far in native cells. We describe single-channel events arising directly from the Caenorhabditis elegans dopamine transporter (DAT-1) as evidenced by DA-induced channel activity blocked by a high-affinity DAT-1 inhibitor, increased channel activity in neurons that overexpress DAT-1, and loss of channels in dat-1 knockout neurons. Our data indicate that authentic transporter channels underlie depolarizing whole-cell currents. Thus, DA transporters not only transport DA but also exhibit a channel mode of conduction that directly modulates membrane potential and neuronal function.     

Functional regulation of large conductance Ca2+-activated K+ channels in vascular diseases

The large conductance Ca²⁺-activated potassium channels, the BK channels, is widely expressed in various tissues and activated in a Ca²⁺- and voltage-dependent manner. The activation of BK channels hyperpolarizes vascular smooth muscle cell membrane potential, resulting in vasodilation. Under pathophysiological conditions, such as diabetes mellitus and hypertension, impaired BK channel function exacerbates vascular vasodilation and leads to organ ischemia. The vascular BK channel is composed of 4 pore-forming subunits, BK-α together with 4 auxiliary subunits: β₁ subunits (BK-β₁) or γ₁ subunits (BK-γ₁). Recent studies have shown that down-regulation of the BK β₁ subunit in diabetes mellitus induced vascular dysfunction; however, the molecular mechanism of these vascular diseases is not well understood. In this review, we summarize the potential mechanisms regarding BK channelopathy and the potential therapeutic targets of BK channels for vascular diseases.     

Inhibition of MAPK stimulates the Ca2+ -dependent big-conductance K channels in cortical collecting duct

The kidney plays a key role in maintaining potassium (K) homeostasis. K excretion is determined by the balance between K secretion and absorption in distal tubule segments such as the connecting tubule and cortical collecting duct. K secretion takes place by K entering principal cells (PC) from blood side through Na+, K+ -ATPase and being secreted into the lumen via both ROMK-like small-conductance K (SK) channels and Ca2+ -activated big-conductance K (BK) channels. K reabsorption occurs by stimulation of apical K/H-ATPase and inhibition of K recycling across the apical membrane in intercalated cells (IC). The role of ROMK channels in K secretion is well documented. However, the importance of BK channels in mediating K secretion is incompletely understood. It has been shown that their activity increases with high tubule flow rate and augmented K intake. However, BK channels have a low open probability and are mainly located in IC, which lack appropriate transporters for effective K secretion. Here we demonstrate that inhibition of ERK and P38 MAPKs stimulates BK channels in both PC and IC in the cortical collecting duct and that changes in K intake modulate their activity. Under control conditions, BK channel activity in PC was low but increased significantly by inhibition of both ERK and P38. Blocking MAPKs also increased channel open probability of BK in IC and thereby it may affect K backflux and net K absorption Thus, modulation of ERK and P38 MAPK activity is involved in controlling net K secretion in the distal nephron.     

Modulation of Two Cloned Potassium Channels by 1-Alkanols Demonstrates Different Cutoffs

It is not known whether alcohols modulate ion channels by directly binding to the channel protein or by perturbing the surrounding membrane lipid. Cutoff describes the phenomenon where the potency of 1-alkanols monotonically increases with alkyl chain length until a loss of efficacy occurs. Determination of the cutoff for a variety of channels can be important, because similar and/or dissimilar cutoffs might yield information regarding the nature of ethanol's site of action. In this study, the two-electrode voltage clamp technique was used to determine the cutoffs for the 1-alkanol potentiation of cloned Ca²⁺-activated-K⁺ (BK) channels and for the inhibition of cloned Shaw2 K⁺ channels, expressed in Xenopus oocytes. Ethanol, butanol, hexanol, and heptanol reversibly enhanced BK currents, whereas octanol and nonanol had no effect. In contrast, Shaw2 currents were potently inhibited by both octanol and decanol, but not by undecanol. Taken together, data demonstrate that the modulation of K⁺ channels by long chain alcohols is channel-specific. Interestingly, ethanol was a less potent activator of BK currents in the intact oocyte in comparison with its effect on this channel in excised membrane patches. The decrease in potency could not be attributed to an ethanol-dependent change in Ca²⁺ influx through endogenous voltage-gated channels, an effect that would alter the concentration of Ca²⁺ available to activate BK channels.     

Inflammatory modulation of calcium-activated potassium channels in canine colonic circular smooth muscle cells

BACKGROUND & AIMS: The characteristics of colonic circular smooth muscle slow waves are altered during inflammation. The aim of this study was to examine whether inflammation modulates the open-state probability of Ca2+-activated K+ (KCa) channels in these cells to contribute to these alterations. METHODS: The experiments were performed on freshly dissociated single smooth muscle cells from the canine colon using standard patch clamp methods. Inflammation was induced by mucosal exposure to ethanol and acetic acid. RESULTS: Inflammation decreased the open-state probability of large-conductance KCa (BK) channels in the cell-attached and excised inside-out configurations. The voltage sensitivity of the channels was also reduced during inflammation. Inflammation had no significant effect on the large, medium, and small conductances or the unitary current levels of channel openings. However, it decreased the maximum number of simultaneous channel openings. The channels were Ca2+-dependent and were blocked by tetraethylammonium and charybdotoxin in normal and inflamed cells. CONCLUSIONS: Inflammation decreases the open-state probability of BK channels. This may partially reverse the decrease in duration and amplitude of slow waves and depolarization of membrane potential seen in inflammation.     

Calcium and small-conductance calcium-activated potassium channels in gonadotropin-releasing hormone neurons before, during, and after puberty

The pubertal increase in GnRH secretion resulting in sexual maturation and reproductive competence is a complex process involving kisspeptin stimulation of GnRH neurons and requiring Ca(2+) and possibly other intracellular messengers. To determine whether the expression of Ca(2+) channels, or small-conductance Ca(2+)-activated K(+) (SK) channels, whose activity reflects cytoplasmic free Ca(2+) concentration, changes at puberty in GnRH neurons, Ca(2+) and SK currents in GnRH neurons were recorded in brain slices of juvenile [postnatal day (P) 10-21], pubertal (P28-P42), and adult (> or =P56) male GnRH-green fluorescent protein transgenic mice using perforated-patch and whole-cell techniques. Ca(2+) currents were inhibited by the Ca(2+) channel blocker Cd(2+) and showed marked heterogeneity but were on average similar in juvenile, pubertal, and adult GnRH neurons. SK currents, which were inhibited by the SK channel blocker apamin and enhanced by the SK and intermediate-conductance Ca(2+)-activated K(+) channel activator 1-ethyl-2-benzimidazolinone, were also on average similar in juvenile, pubertal, and adult GnRH neurons. These findings suggest that whereas Ca(2+) and SK channels may participate in the pubertal increase in GnRH secretion and there may be changes in Ca(2+) or SK channel subtypes, overall Ca(2+) and SK channel expression in GnRH neurons remains relatively constant across pubertal development. Hence, the expected increase in GnRH neuron cytoplasmic free Ca(2+) concentration required for increased GnRH secretion at puberty appears to be due to mechanisms other than altered Ca(2+) or SK channel expression, e.g. increased membrane depolarization and subsequent activation of preexisting Ca(2+) channels after increased excitatory synaptic input.     

Small-conductance calcium-activated potassium channels control excitability and firing dynamics in gonadotropin-releasing hormone (GnRH) neurons

The cellular mechanisms determining the firing patterns of GnRH neurons are presently under intense investigation. In this study, we used GnRH-green fluorescent protein transgenic mice and perforated-patch electrophysiology to examine the role of small conductance calcium-activated potassium (SK) channels in determining the electrical excitability and burst-firing characteristics of adult GnRH neurons. After establishing an appropriate protocol for examining the afterhyperpolarization potential (AHP) currents in GnRH neurons, the highly selective SK channel blocker apamin was used to demonstrate that all GnRH neurons express functional SK channels (35.7 +/- 2.7 pA, mean decay time constant = 2167 msec, apamin IC(50) = 9.6 nm) and that this channel underlies approximately 90% of the AHP in these cells. Current-clamp experiments showed that apamin-sensitive SK channels were tonically active in the majority (74%) of GnRH neurons, with apamin (100 nm) administration resulting in a mean 6.9 +/- 0.5 mV membrane depolarization. Apamin also elevated the firing rate of GnRH neurons, including increased burst frequency and duration in spontaneously bursting cells as well as the ability of GnRH neurons to fire action potentials in response to current injection. In GnRH neurons activated by current injection, apamin significantly enhanced the amplitude of the afterdepolarization potential after a single action potential and eliminated spike frequency adaptation. Together, these studies show that apamin-sensitive SK channels play a key role in restraining GnRH neuron excitability. Through direct modulation of the AHP and indirect actions on the afterdepolarization potential, the SK channel exerts a powerful tonic influence upon the firing dynamics of GnRH neurons.     

Endothelial-derived hyperpolarization factor (EDHF) contributes to PlGF-induced dilation of mesenteric resistance arteries from pregnant rats

The aim of this study was to investigate the cellular mechanism involved in the potent vasodilatory action of PlGF on mesenteric resistance arteries from pregnant rats. PlGF (3 nM) induced a vasodilation of 64 ± 3.8% that was completely abolished by endothelial denudation. Significant dilation (28 ± 4.0%) remained, however, in the presence of nitric oxide synthase and cyclooxygenase inhibition, and was associated with significant reductions in vascular smooth muscle cell calcium. Absence of dilation in potassium-depolarizing solution (30 mM) confirmed its dependence on endothelial-derived hyperpolarization factor. Subsequent studies established that vasodilation was abolished by pharmacologic inhibition of SK(Ca) (apamin) and BK(Ca) (iberiotoxin) but not IK(Ca) (tram-34) potassium channels. In summary, PlGF acts through the release of a combination of endothelium-derived relaxation factors. Based on the results of potassium channel blockade, we suggest that it induces endothelial hyperpolarization via SK(Ca) channel activation; this, in turn, leads to the release of a diffusible mediator that activates vascular smooth muscle BK(Ca) channels, hyperpolarization and vasodilation. This is the first study to identify the mechanism for PlGF/VEGFR-1 resistance artery dilation in the pregnant state, whose attenuation likely contributes to the systemic hypertension characteristic of pre- eclampsia.     

Single-channel analyses of ethanol modulation of neuronal nicotinic acetylcholine receptors

BACKGROUND: We have previously reported that ethanol potentiates the acetylcholine-induced currents of the alpha4beta2 neuronal nicotinic acetylcholine receptors in rat cortical neurons and of those that are stably expressed in human embryonic kidney cells. The potentiation of the maximal currents evoked by high concentrations of acetylcholine suggests that ethanol affects the channel gating. METHODS: We performed single-channel patch-clamp experiments to elucidate the detailed mechanism of ethanol modulation of the alpha4beta2 receptor that is stably expressed in human embryonic kidney cells. RESULTS: At least two conductance states, 40.5 pS and 21.9 pS, were activated by acetylcholine. Acetylcholine at 30 nM predominantly induced the high conductance state currents (85% of total). Ethanol did not affect the single-channel conductance but selectively modulated the high-conductance state currents. The high-conductance state currents exhibited two open time constants. Both time constants were increased by 100 mM ethanol, from 1.9 msec to 2.8 msec and from 9.0 msec to 15.5 msec, respectively. Ethanol also prolonged the burst duration and the open time within burst and increased the probability of channel opening. CONCLUSIONS: These changes in single-channel parameters indicate that ethanol stabilizes the alpha4beta2 receptor-channel in the opening state, explaining how the maximum acetylcholine-induced whole-cell currents are further potentiated by ethanol.     

Altered expression of small-conductance Ca2+-activated K+ (SK3) channels modulates arterial tone and blood pressure

The endothelium is a critical regulator of vascular tone, and dysfunction of the endothelium contributes to numerous cardiovascular pathologies. Recent studies suggest that apamin-sensitive, small-conductance, Ca2+-activated K+ channels may play an important role in active endothelium-dependent vasodilations, and expression of these channels may be altered in disease states characterized by vascular dysfunction. Here, we used a transgenic mouse (SK3T/T) in which SK3 expression levels can be manipulated with dietary doxycycline (DOX) to test the hypothesis that the level of expression of the SK subunit, SK3, in endothelial cells alters arterial function and blood pressure. SK3 protein was elevated in small mesenteric arteries from SK3T/T mice compared with wild-type mice and was greatly suppressed by dietary DOX. SK3 was detected in the endothelium and not in the smooth muscle by immunohistochemistry. In whole-cell patch-clamp experiments, SK currents in endothelial cells from SK3T/T mice were almost completely suppressed by dietary DOX. In intact arteries, SK3 channels contributed to sustained hyperpolarization of the endothelial membrane potential, which was communicated to the arterial smooth muscle. Pressure- and phenylephrine-induced constrictions of SK3T/T arteries were substantially enhanced by treatment with apamin, suppression of SK3 expression with DOX, or removal of the endothelium. In addition, suppression of SK3 expression caused a pronounced and reversible elevation of blood pressure. These results indicate that endothelial SK3 channels exert a profound, tonic, hyperpolarizing influence in resistance arteries and suggest that the level of SK3 channel expression in endothelial cells is a fundamental determinant of vascular tone and blood pressure.