Intracellular Na+ inhibits voltage-dependent N-type Ca2+ channels by a G protein βγ subunit-dependent mechanism

N-type  voltage-dependent  Ca²⁺ channels (N-VDCCs) play important roles in neurotransmitter release and certain postsynaptic phenomena. These channels are modulated by a number of intracellular factors, notably by Gβγ subunits of G proteins, which inhibit N-VDCCs in a voltage-dependent (VD) manner. Here we show that an increase in intracellular Na⁺ concentration inhibits N-VDCCs  in hippocampal pyramidal neurones and in Xenopus oocytes. In acutely dissociated hippocampal neurones, Ba²⁺ current via N-VDCCs was inhibited by Na⁺ influx caused by the activation of NMDA receptor channels. In Xenopus oocytes expressing N-VDCCs, Ba²⁺ currents were inhibited by Na⁺ influx and enhanced by depletion of Na⁺, after incubation in a Na⁺-free extracellular solution. The Na⁺-induced inhibition was accompanied by the development of  VD facilitation, a hallmark of a Gβγ-dependent process. Na⁺-induced regulation of N-VDCCs is Gβγ dependent, as suggested by the blocking of Na⁺ effects by Gβγ scavengers and by excess Gβγ, and may be mediated by the Na⁺-induced dissociation of Gαβγ heterotrimers. N-VDCCs may be novel effectors of Na⁺ion, regulated by the Na⁺ concentration via Gβγ.     

Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+-induced glucagon exocytosis in pancreas

Hormones such as glucagon are secreted by Ca²⁺-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic β-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca²⁺-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca²⁺ sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca²⁺ sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca²⁺-triggered glucagon release from α-cells than insulin secretion from β-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca²⁺-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca²⁺-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic α-cells lacking synaptotagmin-7 exhibited little Ca²⁺-induced exocytosis, whereas all other physiological and morphological parameters of the α-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca²⁺ sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca²⁺ sensors in neurotransmitter, neuropeptide, and hormone secretion.     

Presynaptic N-type and P/Q-type Ca2+ channels mediating synaptic transmission at the calyx of Held of mice

At the nerve terminal, both N- and P/Q-type Ca²⁺ channels mediate synaptic transmission, with their relative contribution varying between synapses and with postnatal age. To clarify functional significance of different presynaptic Ca²⁺ channel subtypes, we recorded N-type and P/Q-type Ca²⁺ currents directly from calyces of Held nerve terminals in α_1A-subunit-deficient mice and wild-type (WT) mice, respectively. The most prominent feature of P/Q-type Ca²⁺ currents was activity-dependent facilitation, which was absent for N-type Ca²⁺ currents. EPSCs mediated by P/Q-type Ca²⁺ currents showed less depression during high-frequency stimulation compared with those mediated by N-type Ca²⁺ currents. In addition, the maximal inhibition by the GABA_B receptor agonist baclofen was greater for EPSCs mediated by N-type channels than for those mediated by P/Q-type channels. These results suggest that the developmental switch of presynaptic Ca²⁺ channels from N- to P/Q-type may serve to increase synaptic efficacy at high frequencies of activity, securing high-fidelity synaptic transmission.     

Small conductance Ca2+-activated K+ channels and calmodulin

Small conductance Ca²⁺-activated K⁺ channels (SK channels) contribute to the long lasting afterhyperpolarization (AHP) that follows an action potential in many central neurones. The biophysical and pharmacological attributes of cloned SK channels strongly suggest that one or more of them underlie the medium component of the AHP that regulates interspike interval and plays an important role in setting tonic firing frequency. The cloned SK channels comprise a distinct subfamily of K⁺ channels. Heterologously expressed SK channels recapitulate the biophysical and pharmacological hallmarks of native SK channels, being gated solely by intracellular Ca²⁺ ions with no voltage dependence to their gating, small unitary conductance values and sensitivity to the bee venom peptide toxin, apamin. Molecular, biochemical and electrophysiological studies have revealed that Ca²⁺ gating in SK channels is due to heteromeric assembly of the SK α pore-forming subunits with calmodulin (CaM). Ca²⁺ binding to the N-terminal E–F hands of CaM is responsible for SK channel gating. Crystallographic studies suggest that SK channels gate as a dimer-of-dimers, and that the physical gate of SK channels resides at or near the selectivity filter of the channels. In addition, Ca²⁺-independent interactions between the SK channel α subunits and CaM are necessary for proper membrane trafficking.     

Local recovery of Ca2+ release in rat ventricular myocytes

Excitation–contraction coupling in the heart depends on the positive feedback process of Ca²⁺-induced Ca²⁺ release (CICR). While CICR provides for robust triggering of Ca²⁺ sparks, the mechanisms underlying their termination remain unknown. At present, it is unclear how a cluster of Ca²⁺ release channels (ryanodine receptors or RyRs) can be made to turn off when their activity is sustained by the Ca²⁺ release itself. We use a novel experimental approach to investigate indirectly this issue by exploring restitution of Ca²⁺ sparks. We exploit the fact that ryanodine can bind, nearly irreversibly, to an RyR subunit (monomer) and increase the open probability of the homotetrameric channel. By applying low concentrations of ryanodine to rat ventricular myocytes, we observe repeated activations of individual Ca²⁺ spark sites. Examination of these repetitive Ca²⁺ sparks reveals that spark amplitude recovers with a time constant of 91 ms whereas the sigmoidal recovery of triggering probability lags behind amplitude recovery by ∼80 ms. We conclude that restitution of Ca²⁺ sparks depends on local refilling of SR stores after depletion and may also depend on another time-dependent process such as recovery from inactivation or a slow conformational change after rebinding of Ca²⁺ to SR regulatory proteins.     

Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic β-cells

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic β-cells by a combination of electrophysiological recordings, intracellular Ca²⁺ ([Ca²⁺]_i) microfluorimetry and insulin release measurements. Addition of palmitate (1 m m , bound to fatty acid-free albumin) to intact islets exposed to 15 m m glucose increased the [Ca²⁺]_i by ∼30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca²⁺]_i and insulin release in the presence of tolbutamide and in islets depolarized by high K⁺ in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K⁺ channels (K_ATP channels). Palmitate (0.5 m m ) augmented exocytosis (measured as an increase in cell capacitance) in single β-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca²⁺ currents rose by ∼25% following addition of 0.5 m m palmitate, an effect that was abolished in the presence of 10 μ m isradipine indicating that the free fatty acid specifically acts on L-type Ca²⁺ channels. The actions of palmitate on exocytosis and Ca²⁺ currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K_ATP channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca²⁺ currents and an increased size of the RRP.     

Role of extracellular Ca2+ in gating of CaV1.2 channels

We examined changes in ionic and gating currents in Ca_V1.2 channels when extracellular Ca²⁺ was reduced from 10 m m to 0.1 μ m . Saturating gating currents decreased by two-thirds ( K _D≈ 40 μ m ) and ionic currents increased 5-fold ( K _D≈ 0.5 μ m ) due to increasing Na⁺ conductance. A biphasic time dependence for the activation of ionic currents was observed at low [Ca²⁺], which appeared to reflect the rapid activation of channels that were not blocked by Ca²⁺ and a slower reversal of Ca²⁺ blockade of the remaining channels. Removal of Ca²⁺ following inactivation of Ca²⁺ currents showed that Na⁺ currents were not affected by Ca²⁺-dependent inactivation. Ca²⁺-dependent inactivation also induced a negative shift of the reversal potential for ionic currents suggesting that inactivation alters channel selectivity. Our findings suggest that activation of Ca²⁺ conductance and Ca²⁺-dependent inactivation depend on extracellular Ca²⁺ and are linked to changes in selectivity.     

Ca2+-activated K+ (BK) channel inactivation contributes to spike broadening during repetitive firing in the rat lateral amygdala

In many neurons, trains of action potentials show frequency-dependent broadening. This broadening results from the voltage-dependent inactivation of K⁺ currents that contribute to action potential repolarisation. In different neuronal cell types these K⁺ currents have been shown to be either slowly inactivating delayed rectifier type currents or rapidly inactivating A-type voltage-gated K⁺ currents. Recent findings show that inactivation of a Ca²⁺-dependent K⁺ current, mediated by large conductance BK-type channels, also contributes to spike broadening. Here, using whole-cell recordings in acute slices, we examine spike broadening in lateral amygdala projection neurons. Spike broadening is frequency dependent and is reversed by brief hyperpolarisations. This broadening is reduced by blockade of voltage-gated Ca²⁺ channels and BK channels. In contrast, broadening is not blocked by high concentrations of 4-aminopyridine (4-AP) or α-dendrotoxin. We conclude that while inactivation of BK-type Ca²⁺-activated K⁺ channels contributes to spike broadening in lateral amygdala neurons, inactivation of another as yet unidentified outward current also plays a role.     

Dual Ca2+ modulation of glycinergic synaptic currents in rodent hypoglossal motoneurones

Glycinergic synapses are implicated in the coordination of reflex responses, sensory signal processing and pain sensation. Their activity is pre- and postsynaptically regulated, although mechanisms are poorly understood. Using patch-clamp recording and Ca²⁺ imaging in hypoglossal motoneurones from rat and mouse brainstem slices, we address here the role of cytoplasmic Ca²⁺ (Ca_i) in glycinergic synapse modulation. Ca²⁺ influx through voltage-gated or NMDA receptor channels caused powerful transient inhibition of glycinergic IPSCs. This effect was accompanied by an increase in both the failure rate and paired-pulse ratio, as well as a decrease in the frequency of mIPSCs, suggesting a presynaptic mechanism of depression. Inhibition was reduced by the cannabinoid receptor antagonist SR141716A and occluded by the agonist WIN55,212-2, indicating involvement of endocannabinoid retrograde signalling. Conversely, in the presence of SR141716A, glycinergic IPSCs were potentiated postsynaptically by glutamate or NMDA, displaying a Ca²⁺-dependent increase in amplitude and decay prolongation. Both presynaptic inhibition and postsynaptic potentiation were completely prevented by strong Ca_i buffering (20 m m BAPTA). Our findings demonstrate two independent mechanisms by which Ca²⁺ modulates glycinergic synaptic transmission: (i) presynaptic inhibition of glycine release and (ii) postsynaptic potentiation of GlyR-mediated responses. This dual Ca²⁺-induced regulation might be important for feedback control of neurotransmission in a variety of glycinergic networks in mammalian nervous systems.     

Interplay between SERCA and sarcolemmal Ca2+ efflux pathways controls spontaneous release of Ca2+ from the sarcoplasmic reticulum in rat ventricular myocytes

Waves of calcium-induced calcium release occur in a variety of cell types and have been implicated in the origin of cardiac arrhythmias. We have investigated the effects of inhibiting the SR Ca²⁺-ATPase (SERCA) with the reversible inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) on the properties of these waves. Cardiac myocytes were voltage clamped at a constant potential between −65 and −40 mV and spontaneous waves evoked by increasing external Ca²⁺ concentration to 4 m m . Application of 100 μ m TBQ decreased the frequency of waves. This was associated with increases of resting [Ca²⁺]_i, the time constant of decay of [Ca²⁺]_i and the integral of the accompanying Na⁺–Ca²⁺ exchange current. There was also a decrease in propagation velocity of the waves. There was an increase of the calculated Ca²⁺ efflux per wave. The SR Ca²⁺ content when a wave was about to propagate decreased to 91.7 ± 3.2%. The period between waves increased in direct proportion to the Ca²⁺ efflux per wave meaning that TBQ had no effect on the Ca²⁺ efflux per unit time. We conclude that (i) decreased wave frequency is not a direct consequence of decreased Ca²⁺ pumping by SERCA between waves but, rather, to more Ca²⁺ loss on each wave; (ii) inhibiting SERCA increases the chance of spontaneous Ca²⁺ release propagating at a given SR content.     

Increased Ca2+ influx through Na+/Ca2+ exchanger during long-term facilitation at crayfish neuromuscular junctions

Intense motor neuron activity induces a long-term facilitation (LTF) of synaptic transmission at crayfish neuromuscular junctions (NMJs) that is accompanied by an increase in the accumulation of presynaptic Ca²⁺ ions during a test train of action potentials. It is natural to assume that the increased Ca²⁺ influx during action potentials is directly responsible for the increased transmitter release in LTF, especially as the magnitudes of LTF and increased Ca²⁺ influx are positively correlated. However, our results indicate that the elevated Ca²⁺ entry occurs through the reverse mode operation of presynaptic Na⁺/Ca²⁺ exchangers that are activated by an LTF-inducing tetanus. Inhibition of Na⁺/Ca²⁺ exchange blocks this additional Ca²⁺ influx without affecting LTF, showing that LTF is not a consequence of the regulation of these transporters and is not directly related to the increase in [Ca²⁺]_i reached during a train of action potentials. Their correlation is probably due to both being induced independently by the strong [Ca²⁺]_i elevation accompanying LTF-inducing stimuli. Our results reveal a new form of regulation of neuronal Na⁺/Ca²⁺ exchange that does not directly alter the strength of synaptic transmission.     

Functional characterization of a Ca2+-activated non-selective cation channel in human atrial cardiomyocytes

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current ( I _ti). I _ti is the result of three different [Ca]_i-sensitive currents: the Na⁺–Ca²⁺ exchange current, a Ca²⁺-activated chloride current and a Ca²⁺-activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized the properties of a Ca²⁺-activated non-selective cation channel (NSC_Ca) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear I–V relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na⁺ and K⁺) and was slightly permeable to Ca²⁺ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the K _d for [Ca²⁺]_i was 20.8 μ m . Channel activity was reduced in the presence of 0.5 m m ATP or 10 μ m glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 m m flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC_Ca channel is a serious candidate to support the delayed after-depolarizations observed in [Ca²⁺] overload and thus may be implicated in the genesis of arrhythmias.     

KCNQ1 is the luminal K+ recycling channel during stimulation of gastric acid secretion

Parietal cell (PC) proton secretion via H⁺/K⁺-ATPase requires apical K⁺ recycling. A variety of K⁺ channels and transporters are expressed in the PC and the molecular nature of the apical K⁺ recycling channel is under debate. This study was undertaken to delineate the exact function of KCNQ1 channels in gastric acid secretion. Acid secretory rates and electrophysiological parameters were determined in gastric mucosae of 7- to 8-day-old KCNQ1^+/+, ^+/– and ^−/− mice. Parietal cell ultrastructure, abundance and gene expression levels were quantified. Glandular structure and PC abundance, and housekeeping gene expression did not differ between the KCNQ1^−/− and ^+/+ mucosae. Microvillar secretory membranes were intact, but basal acid secretion was absent and forskolin-stimulated acid output reduced by ∼90% in KCNQ1^−/− gastric mucosa. Application of a high K⁺ concentration to the luminal membrane restored normal acid secretory rates in the KCNQ1^−/− mucosa. The study demonstrates that the KCNQ1 channel provides K⁺ to the extracellular K⁺ binding site of the H⁺/K⁺-ATPase during acid secretion, and no other gastric K⁺ channel can substitute for this function.     

Selectivity and interactions of Ba2+ and Cs+ with wild-type and mutant TASK1 K+ channels expressed in Xenopus oocytes

The acid-sensitive K⁺ channel, TASK1 is a member of the K⁺-selective tandem-pore domain (K2P) channel family. Like many of the K2P channels, TASK1 is relatively insensitive to conventional channel blockers such as Ba²⁺. In this paper we report the impact of mutating the pore-neighbouring histidine residues, which are involved in pH sensing, on the sensitivity to blockade by Ba²⁺ and Cs⁺; additionally we compare the selectivity of these channels to extracellular K⁺, Na⁺ and Rb⁺. H98D and H98N mutants showed reduced selectivity for K⁺ over both Na⁺ and Rb⁺, and significant permeation of Rb⁺. This enhanced permeability must reflect changes in the structure or flexibility of the selectivity filter. Blockade by Ba²⁺ and Cs⁺ was voltage-dependent, indicating that both ions block within the pore. In 100 m m K⁺, the K _D at 0 mV for Ba²⁺ was 36 ± 10 m m  ( n = 6)  , whilst for Cs⁺ it was 20 ± 6.0 m m  ( n = 5)  . H98D was more sensitive to Ba²⁺ than the wild-type (WT); in addition, the site at which Ba²⁺ appears to bind was altered (WT: δ, 0.64 ± 0.16, n = 6; H98D: δ, 0.16 ± 0.03, n = 5, statistically different from WT; H98N: δ, 0.58 ± 0.09, not statistically different from WT). Thus, the pore-neighbouring residue H98 contributes not only to the pH sensitivity of TASK1, but also to the structure of the conduction pathway.     

Reactive oxygen species reduce myofibrillar Ca2+ sensitivity in fatiguing mouse skeletal muscle at 37°C

The mechanisms of muscle fatigue were studied in small muscle bundles and single fibres isolated from the flexor digitorum brevis of the mouse. Fatigue caused by repeated isometric tetani was accelerated at body temperature (37°C) when compared to room temperature (22°C). The membrane-permeant reactive oxygen species (ROS) scavenger, Tiron (5 m m ), had no effect on the rate of fatigue at 22°C but slowed the rate of fatigue at 37°C to that observed at 22°C. Single fibres were microinjected with indo-1 to measure intracellular calcium. In the accelerated fatigue at 37°C the tetanic [Ca²⁺]_i did not change significantly and the decline of maximum Ca²⁺-activated force was similar to that observed at 22°C. The cause of the greater rate of fatigue at 37°C was a large fall in myofibrillar Ca²⁺ sensitivity. In the presence of Tiron, the large fall in Ca²⁺ sensitivity was abolished and the usual decline in tetanic [Ca²⁺]_i was observed. This study confirms the importance of ROS in fatigue at 37°C and shows that the mechanism of action of ROS is a decline in myofibrillar Ca²⁺ sensitivity.     

Olfactoretinal centrifugal input modulates zebrafish retinal ganglion cell activity: a possible role for dopamine-mediated Ca2+ signalling pathways

The vertebrate retina receives centrifugal input from the brain. In zebrafish, the major centrifugal input originates in the terminal nerve (TN). TN cell bodies are located in the olfactory bulb and ventral telencephalon. The TN projects axons to the retina where they branch in the inner plexiform layer (IPL) and synapse onto several inner retinal cell types, including dopaminergic interplexiform cells (DA-IPCs). This olfactoretinal centrifugal input plays a role in modulating retinal ganglion cell (RGC) activity, probably via dopamine-mediated Ca²⁺ signalling pathways. Normally, dopamine inhibits RGC firing by decreasing the inward Ca²⁺ current. Olfactory stimulation with amino acids decreases dopamine release in the retina, thereby reducing dopaminergic inhibition of RGCs. This model of olfacto-visual integration was directly tested by recording single-unit RGC activity in response to olfactory stimulation in the presence or absence of dopamine receptor blockers. Stimulation of the olfactory neurones increased RGC activity. However, this effect diminished when the dopamine D1 receptors were pharmacologically blocked. In isolated RGCs, the application of dopamine or a dopamine D1 receptor agonist decreased voltage-activated Ca²⁺ current and lowered Ca²⁺ influx. Together, the data suggest that olfactory input has a modulatory effect on RGC firing, and that this effect is mediated by dopamine D1 receptor-coupled Ca²⁺ signalling pathways.     

Effect of dexamethasone on skeletal muscle Na+,K+ pump subunit specific expression and K+ homeostasis during exercise in humans

The effect of dexamethasone on Na⁺,K⁺ pump subunit expression and muscle exchange of K⁺ during exercise in humans was investigated. Nine healthy male subjects completed a randomized double blind placebo controlled protocol, with ingestion of dexamethasone (Dex: 2 × 2 mg per day) or placebo (Pla) for 5 days. Na⁺,K⁺ pump catalytic α1 and α2 subunit expression was ∼17% higher ( P < 0.05) and the structural β1 and β2 subunit expression was ∼6–8% higher ( P < 0.05) after Dex compared with Pla. During one-legged knee-extension for 10 min at low intensity (LI; 18.6 ± 1.0 W), two moderate intensity (51.7 ± 2.4 W) exercise bouts (MI₁: 5 min; 2 min recovery; MI₂: exhaustive) and two high-intensity (71.7 ± 2.5 W) exercise bouts (HI₁: 1 min 40 s; 2 min recovery; HI₂: exhaustive), femoral venous K⁺ was lower ( P < 0.05) in Dex compared with Pla. Thigh K⁺ release was lower ( P < 0.05) in Dex compared with Pla in LI and MI, but not in HI. Time to exhaustion in MI₂ tended to improve (393 ± 50 s versus 294 ± 41 s; P = 0.07) in Dex compared with Pla, whereas no difference was detected in HI₂ (106 ± 10 s versus 108 ± 9 s). The results indicate that an increased Na⁺,K⁺ pump expression per se is of importance for thigh K⁺ reuptake at the onset of low and moderate intensity exercise, but less important during high intensity exercise.     

Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium

In the mammalian heart, Ca²⁺-independent, depolarization-activated potassium (K⁺) currents contribute importantly to shaping the waveforms of action potentials, and several distinct types of voltage-gated K⁺ currents that subserve this role have been characterized. In most cardiac cells, transient outward currents, I _to,f and/or I _to,s, and several components of delayed reactivation, including I _Kr, I _Ks, I _Kur and I _K,slow, are expressed. Nevertheless, there are species, as well as cell-type and regional, differences in the expression patterns of these currents, and these differences are manifested as variations in action potential waveforms. A large number of voltage-gated K⁺ channel pore-forming (α) and accessory (β, minK, MiRP) subunits have been cloned from or shown to be expressed in heart, and a variety of experimental approaches are being exploited in vitro and in vivo to define the relationship(s) between these subunits and functional voltage-gated cardiac K⁺ channels. Considerable progress has been made in defining these relationships recently, and it is now clear that distinct molecular entities underlie the various electrophysiologically distinct repolarizing K⁺ currents (i.e. I _to,f, I _to,s, I _Kr, I _Ks, I _Kur, I _K,slow, etc.) in myocyardial cells.     

Freshly isolated bovine coronary endothelial cells do not express the BKca channel gene

Recent reports have suggested that different types of Ca²⁺-activated K⁺ channels may be selectively expressed either in the vascular endothelial cells (ECs) or smooth muscle cells (SMCs) of a single artery. In this study, we directly compared mRNA, protein and functional expression of the high-conductance Ca²⁺-activated K⁺ (BK_Ca) channel between freshly isolated ECs and SMCs from bovine coronary arteries. Fresh ECs and SMCs were enzymatically isolated, and their separation verified by immunofluorescent detection of α-actin and platelet/endothelium cell adhesion molecule (PECAM) proteins, respectively. Subsequently, studies using a sequence-specific antibody directed against the pore-forming α-subunit of the BK_Ca channel only detected its expression in the SMCs, whereas PECAM-positive ECs were devoid of the α-subunit protein. Additionally, multicell RT-PCR performed using cDNA derived from either SMCs or ECs only detected mRNA encoding the BK_Caα-subunit in the SMCs. Finally, whole-cell recordings of outward K⁺ current detected a prominent iberiotoxin-sensitive BK_Ca current in SMCs that was absent in ECs, and the BK_Ca channel opener NS 1619 only enhanced K⁺ current in the SMCs. Thus, bovine coronary SMCs densely express BK_Ca channels whereas adjacent ECs in the same artery appear to lack the expression of the BK_Ca channel gene. These findings indicate a cell-specific distribution of Ca²⁺-activated K⁺ channels in SMCs and ECs from a single arterial site.