Calcium-dependent Potassium Channels Research Articles

Caulerpenyne, a toxin from the seaweed Caulerpa taxifolia, depresses afterhyperpolarization in invertebrate neurons

The massive invasion of the Mediterranean Sea by the tropical seaweed Caulerpa taxifolia (Vahl) C. Agardh has stimulated several investigations in order to test the environmental risk from an ecotoxicological point of view. The studies carried out on various experimental models have shown that caulerpenyne, the major metabolite synthesized by the seaweed, affects several cellular and molecular targets. In addition, neurological disorders have been reported in patients who accidentally ate C. taxifolia, but no evidence about the potential effects of the seaweed and of its metabolites on nerve cells were up to now available. Herein we describe that caulerpenyne modifies the electrical properties of touch mechanosensory cells of the leech Hirudo medicinalis. The physiological firing of these cells causes an afterhyperpolarization that is mainly due to the activity of the Na +/K +-ATPase and to a lesser extent to a calcium-dependent potassium current. Caulerpenyne depressed this afterhyperpolarization; the effect was dose-dependent and partially reversible. Experiments have been carried out in order to understand the mechanism through which caulerpenyne reduced the afterhyperpolarization. The action of the biotoxin has been tested in the presence of pharmacological blockers of calcium-dependent potassium channels such as cadmium and apamin. In these experimental conditions, caulerpenyne still reduced the residual afterhyperpolarization, suggesting a direct effect of the toxin on the Na +/K +-ATPase. In order to test this hypothesis, we have performed experiments where the Na +/K +-ATPase was activated by the intracellular injection of sodium and where also its basal activity was modified as well. From the data collected we suggest that caulerpenyne inhibits both the basal and the sodium-induced activity of the Na +/K +-ATPase in leech touch neurons.

Arachidonate dilates basilar artery by lipoxygenase-dependent mechanism and activation of K(+) channels.

Dilatation of cerebral arterioles in response to arachidonic acid is dependent on activity of cyclooxygenase. In this study, we examined mechanisms that mediate dilatation of the basilar artery in response to arachidonate. Diameter of the basilar artery (baseline diameter = 216 +/- 7 micrometer) (means +/- SE) was measured using a cranial window in anesthetized rats. Arachidonic acid (10 and 100 microM) produced concentration-dependent vasodilatation that was not inhibited by indomethacin (10 mg/kg iv) or N(G)-nitro-L-arginine (100 microM) but was inhibited markedly by baicalein (10 micrometerM) or nordihydroguaiaretic acid (NDGA; 10 microM), inhibitors of the lipoxygenase pathway. Dilatation of the basilar artery was also inhibited markedly by tetraethylammonium ion (TEA; 1 mM) or iberiotoxin (50 nM), inhibitors of calcium-dependent potassium channels. For example, 10 microM arachidonate dilated the basilar artery by 19 +/- 7 and 1 +/- 1% in the absence and presence of iberiotoxin, respectively. Measurements of membrane potential indicated that arachidonate produced hyperpolarization of the basilar artery that was blocked completely by TEA. Incubation with [(3)H]arachidonic acid followed by reverse-phase and chiral HPLC indicated that the basilar artery produces relatively small quantities of prostanoids but large quantities of 12(S)-hydroxyeicosatetraenoic acid (12-S-HETE), a lipoxygenase product. Moreover, the production of 12-HETE was inhibited by baicalein or NDGA. These findings suggest that dilatation of the basilar artery in response to arachidonate is mediated by a product(s) of the lipoxygenase pathway, with activation of calcium-dependent potassium channels and hyperpolarization of vascular muscle.

Ca(2+)-activated K(+) channels modulate basal and E(2)beta-induced rises in uterine blood flow in ovine pregnancy.

Uterine blood flow (UBF) increases >30-fold during ovine pregnancy. During the last trimester, this reflects vasodilation, which may be due to placentally derived estrogens. In nonpregnant ewes, estradiol-17 beta (E(2)beta) increases UBF >10-fold by activating nitric oxide synthase and large conductance calcium-dependent potassium channels (BK(Ca)). To determine whether BK(Ca) channels modulate basal and E(2)beta-induced increases in UBF, studies were performed in near-term pregnant ewes with uterine artery flow probes and catheters for intra-arterial infusions of tetraethylammonium (TEA), a selective BK(Ca) channel antagonist at <1 mM, in the absence or presence of E(2)beta (1 microg/kg iv). Uterine arteries were collected to measure BK(Ca) channel mRNA. TEA (0.15 mM) decreased basal UBF (P < 0.0001) 40 +/- 8% and 55 +/- 7% (n = 11) at 60 and 90 min, respectively, and increased resistance 175 +/- 48% without affecting (P > 0.1) mean arterial pressure (MAP), heart rate, or contralateral UBF. Systemic E(2)beta increased UBF 30 +/- 6% and heart rate 13 +/- 1% (P < or = 0.0001, n = 13) without altering MAP. Local TEA (0.15 mM) inhibited E(2)beta-induced increases in UBF without affecting increases in heart rate (10 +/- 4%; P = 0.006). BK(Ca) channel mRNA was present in uterine artery myocytes from pregnant and nonpregnant ewes. Exponential increases in ovine UBF in late pregnancy may reflect BK(Ca) channel activation, which may be mediated by placentally derived estrogens.

The effect of proteolytic enzymes on the α9-nicotinic receptor-mediated response in isolated frog vestibular hair cells

In frog vestibular organs, efferent neurons exclusively innervate type II hair cells. Acetylcholine, the predominant efferent transmitter, acting on acetylcholine receptors of these hair cells ultimately inhibits and/or facilitates vestibular afferent firing. A coupling between α9-nicotinic acetylcholine receptors (α9nAChR) and apamin-sensitive, small-conductance, calcium-dependent potassium channels (SK) is thought to drive the inhibition by hyperpolarizing hair cells thereby decreasing their release of transmitter onto afferents. The presence of α9nAChR in these cells was demonstrated using pharmacological, immunocytochemical, and molecular biological techniques. However, fewer than 10% of saccular hair cells dissociated using protease VIII, protease XXIV, or papain responded to acetylcholine during perforated-patch clamp recordings. When present, these responses were invariably transient, small in amplitude, and difficult to characterize. In contrast, the majority of saccular hair cells (∼90%) dissociated using trypsin consistently responded to acetylcholine with an increase in outward current and concomitant hyperpolarization. In agreement with α9nAChR pharmacology obtained in other hair cells, the acetylcholine response in saccular hair cells was reversibly antagonized by strychnine, curare, tetraethylammonium, and apamin. Brief perfusions with either protease or papain permanently abolished the α9-nicotinic response in isolated saccular hair cells. These enzymes when inactivated became completely ineffective at abolishing the α9-nicotinic response, suggesting an enzymatic interaction with the α9nAChR and/or downstream effector. The mechanism by which these enzymes render saccular hair cells unresponsive to acetylcholine remains unknown, but it most likely involves proteolysis of α9nAChR, SK, or both.

Differential expression of voltage-activated calcium channels in functionally distinct brainstem motoneurons controlling airway and extraocular muscles

Voltage-activated calcium channels are important in controlling motoneuron (MN) excitability and repetitive firing behaviour through their effects on calcium-dependent potassium channels, and the after-hyperpolarization (AHP), in particular the medium AHP (mAHP). Differential expression of voltage-activated calcium channels may therefore be important in establishing the task-specific firing properties of functionally distinct motoneuron pools. Differential expression of these channels may also be involved in the variable susceptibility of MNs to disruptions in calcium homeostasis, which are implicated in the pathology of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS). To further explore the expression of calcium channels in brainstem MNs and the possibility that differential expression of calcium channels predisposes some MNs to degeneration, we compared voltage-activated calcium channels in hypoglossal (XII) and oculomotor (III) MNs, that differ not only in their firing behaviour, but in their vulnerability to degeneration in ALS. XII MNs, which innervate the genioglossus muscle of the tongue and degenerate in ALS, produce rhythmic bursts of action potentials during inspiration that last ~200–400 ms. Discharge frequency peaks between 25–50 Hz [1]. Oculomotor motoneurons, which innervate extraocular muscles and are resistant to degeneration in ALS, can produce sustained firing rates of 300 Hz during fixation and peak discharge rates of 600 Hz for up to 25 ms during saccades [2]. Voltage-activated calcium channel expression was compared between XII and III MNs electrophysiologically in neonatal rat brain slices using whole-cell patch-clamp recording techniques, and immunohistochemically using antibodies to different channel subtypes. Current densities of low voltage-activated (LVA) calcium currents were similar in XII (-5.9 ± 1.1 pA/pF, n = 9) and III (-4.6 ± 0.8 pA/pF, n = 13) MNs. Immunolabelling for the α1 G subunit of the LVA calcium channel was also similar between MN pools. In contrast, high voltage-activated (HVA) calcium current density was two-fold greater in XII (-38.2 ± 3.0 pA/pF, n = 33) compared with III (-19.5 ± 1.4 pA/pF, n = 40) MNs. Use of HVA calcium channel antagonists (nimodipine and nifedipine for L-type; ω-agatoxin-TK for P/Q-type; ω-conotoxin-GVIA for N-type) revealed that the majority of this difference reflected greater P/Q-type currents in XII MNs (XII: -15.4 ± 0.9 pA/pF, n = 6; III: -4.4 ± 1.2 pA/pF, n = 12). Immunohistochemical analysis of P/Q channel expression supported greater expression in XII MNs. N-type currents were not significantly different in XII (-4.3 ± 1.2 pA/pF, n = 9) and III MNs (-8.0 ± 1.7 pA/pF; n = 11). L-type currents, defined by nimodipine or nifedipine sensitivity, were not detected. These data suggest that differential expression of voltage-activated calcium channels may not contribute to the distinct firing properties of XII and III MNs since activation of the mAHP is primarily associated with calcium flux through N-type channels [3] and these channels do not differ between XII and III MNs. However, our data do support the possibility that greater expression of HVA calcium channels may predispose XII MNs to degeneration during periods of metabolic stress as seen in ALS. The greater density of P/Q-type HVA calcium channels in XII MNs would allow more calcium to enter MNs upon depolarisation and increase the likelihood of interaction with calcium channel antibodies found in some ALS patients.

Intrinsic Membrane Properties Underlying Spontaneous Tonic Firing in Neostriatal Cholinergic Interneurons

Neostriatal cholinergic interneurons produce spontaneous tonic firing in the absence of synaptic input. Perforated patch recording and whole-cell recording combined with calcium imaging were used in vitro to identify the intrinsic membrane properties underlying endogenous excitability. Spontaneous firing was driven by the combined action of a sodium current and the hyperpolarization-activated cation current (I(h)), which together ensured that there was no zero current point in the subthreshold voltage range. Blockade of sodium channels or I(h) established a stable subthreshold resting membrane potential. A tetrodotoxin-sensitive region of negative slope conductance was observed between approximately -60 mV and threshold (approximately -50 mV) and the h-current was activated at all subthreshold voltages. Calcium imaging experiments revealed that there was minimal calcium influx at subthreshold membrane potentials but that action potentials produced elevations of calcium in both the soma and dendrites. Spike-triggered calcium entry shaped the falling phase of the action potential waveform and activated calcium-dependent potassium channels. Blockade of big-conductance channels caused spike broadening. Application of apamin, which blocks small-conductance channels, abolished the slow spike afterhyperpolarization (AHP) and caused a transition to burst firing. In the absence of synaptic input, a range of tonic firing patterns are observed, suggesting that the characteristic spike sequences described for tonically active cholinergic neurons (TANs) recorded in vivo are intrinsic in origin. The pivotal role of the AHP in regulating spike patterning indicates that burst firing of TANs in vivo could arise from direct or indirect modulation of the AHP without requiring phasic synaptic input.

Analogue pattern matching in a dendritic spine model based on phosphorylation of potassium channels

Modification of potassium channels by protein phosphorylation has been shown to play a role in learning and memory. If such memory storage machinery were part of dendritic spines, then a set of spines could act as an ‘analogue pattern matching’ device by learning a repeatedly presented pattern of synaptic activation. In this study, the plausibility of such analogue pattern matching is investigated in a detailed circuit model of a set of spines attached to a dendritic branch. Each spine head contains an AMPA synaptic channel in parallel with a calcium-dependent potassium channel whose sensitivity depends on its phosphorylation state. Repeated presentation of synaptic activity results in calcium activation of protein kinases and subsequent channel phosphorylation. Simulations demonstrate that signal strength is greatest when the synaptic input pattern is equal to the previously learned pattern, and smaller when components of the synaptic input pattern are either smaller or larger than corresponding components of the previously learned pattern. Therefore, our results indicate that dendritic spines may act as an analogue pattern matching device, and suggest that modulation of potassium channels by protein kinases may mediate neuronal pattern recognition.

An evaluation of potassium ions as endothelium-derived hyperpolarizing factor in porcine coronary arteries.

In the rat hepatic artery, the endothelium-derived hyperpolarizing factor (EDHF) was identified as potassium. Potassium hyperpolarizes the smooth muscles by gating inward rectified potassium channels and by activating the sodium-potassium adenosine triphosphatase (Na(+)-K(+)ATPase). Our goal was to examine whether potassium could explain the EDHF in porcine coronary arteries. On coronary strips, the inhibition of calcium-dependent potassium channels with 100 nM apamin plus 100 microM charibdotoxin inhibited the endothelium-dependent relaxations, produced by 10 nM substance P and 300 nM bradykinin and resistant to nitro-L-arginine and indomethacin. The scavenging of potassium with 2 mM Kryptofix 2.2.2 abolished the endothelium-dependent relaxations produced by the kinins and resistant to nitro-L-arginine and indomethacin. Forty microM 18alpha glycyrrethinic acid or 50 microM palmitoleic acid, both uncoupling agents, did not inhibit these kinin relaxations. Therefore, EDHF does not result from an electrotonic spreading of an endothelial hyperpolarization. Barium (0.3 nM) did not inhibit the kinin relaxations resistant to nitro-L-arginine and indomethacin. Therefore, EDHF does not result from the activation of inward rectified potassium channels. Five hundred nM ouabain abolished the endothelium-dependent relaxations resistant to nitro-L-arginine and indomethacin without inhibiting the endothelium-derived NO relaxation. The perifusion of a medium supplemented with potassium depolarized and contracted a coronary strip; however, the short application of potassium hyperpolarized the smooth muscles. These results are compatible with the concept that, in porcine coronary artery, the EDHF is potassium released by the endothelial cells and that this ion hyperpolarizes and relaxes the smooth muscles by activating the Na(+)-K(+)ATPase.

Vasoregulation by the beta1 subunit of the calcium-activated potassium channel.

Small arteries exhibit tone, a partially contracted state that is an important determinant of blood pressure. In arterial smooth muscle cells, intracellular calcium paradoxically controls both contraction and relaxation. The mechanisms by which calcium can differentially regulate diverse physiological responses within a single cell remain unresolved. Calcium-dependent relaxation is mediated by local calcium release from the sarcoplasmic reticulum. These 'calcium sparks' activate calcium-dependent potassium (BK) channels comprised of alpha and beta1 subunits. Here we show that targeted deletion of the gene for the beta1 subunit leads to a decrease in the calcium sensitivity of BK channels, a reduction in functional coupling of calcium sparks to BK channel activation, and increases in arterial tone and blood pressure. The beta1 subunit of the BK channel, by tuning the channel's calcium sensitivity, is a key molecular component in translating calcium signals to the central physiological function of vasoregulation.

Inhibition of small conductance K+-channels attenuated melatonin-induced relaxation of serotonin-contracted rat gastric fundus

The aim of this study was to investigate the effects of melatonin on rat gastric fundus smooth muscle. Melatonin (10-4 to10-3 M) had no effect on the basal tone of gastric smooth muscle. After precontraction with carbachol (10-6 M) or serotonin (10-7 M), melatonin caused a concentration dependent inhibitory action. The half maximal effect on serotonin-induced contraction was found with 1.12 beta 0.86 beta 10-5 M of melatonin. Increasing concentrations of melatonin (10-5 to 10-3 M) resulted in a right shift of the serotonin concentration response curve (10-10 to10-5 M). This inhibitory effect of melatonin was partially blocked in the presence of apamin (10-10 to 10-7 M), a specific blocker of the small conductance calcium-dependent potassium channel, but not in the presence of other potassium channel blockers like charybdotoxin (10-8 M), glibenclamide (10-5 M), or tetraethylammonium (ODQ, 10-4 M). The inhibitory effect was not changed in the presence of the neuronal blocker tetrodotoxin (10-6 M), the selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3 × 10-5 M), the nitric-oxide synthase inhibitor N-nitro-L-arginine (3 × 10-4 M), or the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (10-4 M), suggesting that neither the purinergic, nitrergic, nor guanylate cyclase pathways were involved. We further investigated inhibitory responses to electrical field stimulation (EFS) at different frequencies under non-adrenergic, non-cholinergic (NANC) conditions on a serotonin-induced contraction in the presence of melatonin (10-5 to 10-4 M). Melatonin significantly reduced these inhibitory NANC responses in higher (8-32 Hz), but not lower (05-4 Hz), frequencies (16 Hz without melatonin, 103 ± 6.3%; melatonin 10-5 M, 80.4 ± 7.5%; melatonin 10-4 M, 39.1 ± 17.1%). Melatonin had no effect on contractile responses induced by EFS under basal tone. These results demonstrate that the inhibitory effect of melatonin in rat gastric fundus smooth muscle is apamin sensitive, but is not affected by other potassium channel blockers. This suggests that melatonin may be another transmitter candidate for the apamin sensitive responses within the gastrointestinal tract.Key words: melatonin, smooth muscle, apamin, NANC-inhibition, gastric, potassium channels.

Improvement by phosphoramidon of damaged endothelial function in porcine coronary artery

Background. The bradykinin (BK)-induced endothelium-dependent relaxation is impaired in the presence of elevated potassium concentration enhancing the vasospastic tendency of large coronary arteries. Inhibition of the angiotensin-converting enzyme responsible for bradykinin degradation was found to enhance the endothelium-dependent relaxation by BK. The aim of the present study was to investigate the effect of phosphoramidon, known to inhibit a BK-metabolizing neutral endopeptidase enzyme, on relaxation of porcine-isolated coronary artery in depolarizing solution. Methods. Endothelium intact porcine coronary artery rings were studied in organ chambers. The rings were isometrically contracted with potassium chloride (30 mmol/L) and the response to BK (1 to 1,000 nmol/L)-induced relaxation was investigated in the presence of nitric oxide synthase inhibitor N ω-nitro- L-arginine (300 μmol/L) alone and in combination with the cyclooxygenase inhibitor indomethacin (10 μmol/L), and that of the inhibitor of calcium-dependent potassium channels tetraethylammonium (7 mmol/L). Under these conditions, phosphoramidon (10 μmol/L), an inhibitor of a neutral endopeptidase enzyme (EC.3.4.24.11.), which is responsible for the degradation of BK, was used to enhance the endothelium-dependent relaxation. Results. Phosphoramidon potentiated the maximum vasorelaxant effect of BK in N ω-nitro- L-arginine (control 26.6% ± 10.86% versus phosphoramidon 49.05% ± 4.52%; n = 6, p < 0.05) or in N ω-nitro- L-arginine + indomethacin-pretreated rings (control 20.7% ± 9.92% versus phosphoramidon 42.0% ± 12.26%; n = 5, p < 0.05) and this increased vasodilation was not modified by tetraethylammonium. Conclusions. In the present study phosphoramidon potentiated the effect of BK in the absence of nitric oxide and prostaglandins in porcine-isolated coronary artery. This effect did not depend on tetraethylammonium-sensitive potassium channels. Phosphoramidon may be a useful pharmacologic tool for preserving the vasorelaxing capacity of coronary arteries after cardioplegia.

An Earful of AChR α9

Even before hearing becomes active, there is a transient cholinergic innervation in the developing mammalian inner ear. The physiological significance of these synapses has never been demonstrated. Glowatzki and Fuchs show that the novel neuronal nicotinic acetylcholine receptor α9 is functionally present in neonatal cochlear inner hair cells and behaves like a true acetylcholine receptor. Its stimulation results in the activation of a fast, calcium-dependent potassium channel. Acetylcholine thus inhibits afferent activity and acts to impose rhythmicity onto the immature auditory pathway. Glowatzki, E., and Fuchs, P.A. (2000) Cholinergic synaptic inhibition of inner hair cells in the neonatal mammalian cochlea. Science 288 : 2366-2368. [Abstract] [Full Text]

Calcium-activated potassium channels and nitric oxide coregulate estrogen-induced vasodilation.

Nitric oxide synthase (NOS) contributes to estradiol-17beta (E(2)beta)-induced uterine vasodilation, but additional mechanisms are involved, and the cellular pathways remain unclear. We determined if 1) uterine artery myocytes express potassium channels, 2) E(2)beta activates these channels, and 3) channel blockade plus NOS inhibition alters E(2)beta-induced uterine vasodilation. Studies of cell-attached patches identified a 107 +/- 7 pS calcium-dependent potassium channel (BK(Ca)) in uterine artery myocytes that rapidly increased single-channel open probability 70-fold (P < 0.05) after exposure to 100 nM E(2)beta through an apparent cGMP-dependent mechanism. In ovariectomized nonpregnant ewes (n = 11) with uterine artery flow probes and catheters, local BK(Ca) blockade with tetraethylammonium (TEA; 0.05-0.6 mM) dose dependently inhibited E(2)beta-induced uterine vasodilation (n = 37, R = 0.77, P < 0.0001), with maximum inhibition averaging 67 +/- 11%. Mean arterial pressure (MAP) and E(2)beta-induced increases (P </= 0.001) in heart rate (13%) and contralateral uterine blood flow (UBF, approximately 5-fold) were unaffected. Local NOS inhibition plus BK(Ca) blockade, using submaximal doses of nitro-L-arginine methyl ester (5 mg/ml) and TEA (0.3 mM), did not alter basal UBF but completely inhibited ipsilateral E(2)beta-induced uterine vasodilation without affecting MAP and E(2)beta-induced increases in contralateral UBF and heart rate. Acute E(2)beta-mediated uterine vasodilation involves rapid activation of uterine artery BK(Ca) and NOS, and the pathway for their interaction appears to include activation of guanylyl cyclase.

Cholinergic synaptic inhibition of inner hair cells in the neonatal mammalian cochlea.

Efferent feedback onto sensory organs provides a means to modulate input to the central nervous system. In the developing mammalian cochlea, inner hair cells are transiently innervated by efferent fibers, even before sensory function begins. Here, we show that neonatal inner hair cells are inhibited by cholinergic synaptic input before the onset of hearing. The synaptic currents, as well as the inner hair cell's response to acetylcholine, are mediated by a nicotinic (alpha9-containing) receptor and result in the activation of small-conductance calcium-dependent potassium channels.

Development of potassium conductances in perinatal rat phrenic motoneurons.

Prior to the inception of inspiratory synaptic drive transmission from medullary respiratory centers, rat phrenic motoneurons (PMNs) have action potential and repetitive firing characteristics typical of immature embryonic motoneurons. During the period spanning from when respiratory bulbospinal and segmental afferent synaptic connections are formed at embryonic day 17 (E17) through to birth (gestational period is approximately 21 days), a pronounced transformation of PMN electrophysiological properties occurs. In this study, we test the hypothesis that the elaboration of action potential afterpotentials and the resulting changes in repetitive firing properties are due in large part to developmental changes in PMN potassium conductances. Ionic conductances were measured via whole cell patch recordings using a cervical slice-phrenic nerve preparation isolated from perinatal rats. Voltage- and current-clamp recordings revealed that PMNs expressed outward rectifier (I(KV)) and A-type potassium currents that regulated PMN action potential and repetitive firing properties throughout the perinatal period. There was an age-dependent leftward shift in the activation voltage and a decrease in the time-to-peak of I(KV) during the period from E16 through to birth. The most dramatic change during the perinatal period was the increase in calcium-activated potassium currents after the inception of inspiratory drive transmission at E17. Block of the maxi-type calcium-dependent potassium conductance caused a significant increase in action potential duration and a suppression of the fast afterhyperpolarizing potential. Block of the small conductance calcium-dependent potassium channels resulted in a marked suppression of the medium afterhyperpolarizing potential and an increase in the repetitive firing frequency. In conclusion, the increase in calcium-mediated potassium conductances are in large part responsible for the marked transformation in action potential shape and firing properties of PMNs from the time between the inception of fetal respiratory drive transmission and birth.

A Novel Nervous System β Subunit that Downregulates Human Large Conductance Calcium-Dependent Potassium Channels

The pore-forming alpha subunits of many ion channels are associated with auxiliary subunits that influence channel expression, targeting, and function. Several different auxiliary (beta) subunits for large conductance calcium-dependent potassium channels of the Slowpoke family have been reported, but none of these beta subunits is expressed extensively in the nervous system. We describe here the cloning and functional characterization of a novel Slowpoke beta4 auxiliary subunit in human and mouse, which exhibits only limited sequence homology with other beta subunits. This beta4 subunit coimmunoprecipitates with human and mouse Slowpoke. beta4 is expressed highly in human and monkey brain in a pattern that overlaps strikingly with Slowpoke alpha subunit, but in contrast to other Slowpoke beta subunits, it is expressed little (if at all) outside the nervous system. Also in contrast to other beta subunits, beta4 downregulates Slowpoke channel activity by shifting its activation range to more depolarized voltages and slowing its activation kinetics. beta4 may be important for the critical roles played by Slowpoke channels in the regulation of neuronal excitability and neurotransmitter release.

Role of nucleotide cyclases in the inhibition of pregnant rat uterine contractions by the openers of potassium channels

Objective: Our objective was to study the involvement of adenylate and guanylate cylases in spontaneous uterine contractions and inhibition induced by the opening of potassium channels. Study Design: Uterine rings from rats at mid and term gestation and from rats at term gestation in labor were suspended in organ chambers for isometric tension recording. Concentration-response relationships to an opener of adenosine triphosphate–dependent potassium channels, levcromakalim, or to an opener of calcium-dependent potassium channels, NS 1619, were studied in the absence and presence of inhibitors of adenylate cyclase (MDL 12330 A, 2 × 10 –5 mol/L; SQ 22536, 10 –4 mol/L) or guanylate cyclase (LY 83583,3 × 10 –6 mol/L). Results: MDL 12330 A and SQ 22536 accentuated contractions in rings from rats at mid gestation but not at term gestation or at term gestation in labor. LY 83583 inhibited contractions in the rings from all 3 groups. Levcromakalim was equally effective in inhibiting contractions of rings from all 3 groups. MDL 12330 A, but not SQ 22536, decreased sensitivity and maximal inhibition induced by levcromakalim (term gestation greater than mid gestation greater than term gestation in labor). LY 83583 decreased the sensitivity to and maximal inhibition induced by levcromakalim in rings from pregnant rats at mid gestation. NS 1619 attenuated contraction of rings from rats at mid gestation and, to a lesser extent, at term gestation but accentuated contractions in rings from animals at term gestation in labor. MDL 12330 A, but not SQ 22536 or LY 83583, attenuated the changes induced by NS 1619 in rings from all 3 groups. Conclusions: (1) The influence of nucleotide cyclases on basal uterine contractility depends on gestational age. (2) The inhibition of uterine contractions that results from the opening of calcium-dependent potassium channels depends on adenylate cyclase, whereas that of adenosine triphosphate–dependent potassium channels depends on both adenylate and guanylate cyclases. 3. Activation of adenosine triphosphate–dependent potassium channels is more efficient than activation of calcium-dependent potassium channels. 4. The inhibition induced by calcium-dependent potassium channel openers, but not adenosine triphosphate–dependent potassium channel openers, decreases as pregnancy progresses, and at delivery the activation of spontaneous contractile activity is evident. (Am J Obstet Gynecol 2000;182:913-8.)

Dehydration of mature and immature sickle red blood cells during fast oxygenation/deoxygenation cycles: role of KCl cotransport and extracellular calcium

AbstractSickle red blood cells (RBC) become dehydrated as a consequence of potassium loss. This process depends at least partly on deoxygenation and may be influenced by the presence of oxygenation/deoxygenation cycles and the frequency of cycling. In this study, sickle RBC were subjected to approximately 180 oxygenation/deoxygenation cycles during 4 hours to evaluate RBC dehydration with cycle periods more similar to in vivo cycles than those in previous studies. A continuous-flow, steady-state apparatus circulated a dilute RBC suspension through gas-permeable silicone tubing with segments that were exposed to either nitrogen or ambient oxygen. The percentage of sickling and partial pressure of oxygen were measured by means of sampling ports in the deoxygenation and oxygenation regions. The density increase (dehydration) of young (transferrin receptor-positive) and mature (transferrin receptor-negative) RBC and the requirements for calcium and chloride were evaluated. Density increase correlated with the percentage of sickled cells at the deoxygenation sampling port and was observed only in the presence of calcium, thereby implicating the calcium-dependent potassium channel (Gardos pathway). Density increase was not dependent on the presence of chloride, making it unlikely that KCl cotransport was an important pathway under these conditions.

Adenylate cyclase an potassium channels are involved in forskolin- and 1,9-dideoxyforskolin-induced inhibition of pregnant rat uterus contractility

Objective:We sought to study the contribution of potassium channels in the effect of forskolin and 1,9-dideoxyforskolin on uterine contractility in the pregnant rat. Study Design:Rings taken from the middle portions of uterine horns from rats at 16 days of gestation were positioned in organ chambers containing physiologic salt solution bubbled with 5% carbon dioxide in air (37°C, pH −7.4) for isometric tension recording under 2 g passive tension. The effects of cumulative concentrations of forskolin and 1,9-dideoxyforskolin in the absence or presence of an adenylate cyclase inhibitor (MDL-12,330A, 10−5 mol/L), a nonselective potassium channel blocker (tetrabutylammonium, 10−4 mol/L), or an adenosine triphosphate-dependent potassium channel blocker (glibenclamide 10−5 mol/L) were studied. Results:Both forskolin and, to a lesser extent, 1,9-dideoxyforskolin inhibit uterine contractions. Tetrabutylammonium, glibenclamide, and MDL-12,330A attenuated the effects of forskolin, whereas glibenclamide was less effective against 1,9-dideoxyforskolin. Conclusions:Activation of adenylate cyclases, as well as adenosine triphosphate-dependent potassium channels and, to a greater extent, calcium-dependent potassium channels, is involved in the inhibitory effect of forskolin in uterine rings from rats at 16 days of gestation. Inhibition of uterine contractions by 1,9-dideoxyforskolin is less than that by forskolin and involves activation of adenylate cyclase and calcium-dependent potassium channels. Whether activation of guanylate cyclase is involved in the effect of the agents on calcium-dependent potassium channels needs further investigation. 1,9-Dideoxyforskolin is not an inactive isomer of forskolin in rat uterine rings.

Inhibitory neural pathway regulating gastric emptying in rats

The relaxation of the pylorus is one of the most important factors for promoting gastric emptying. However, the role of inhibitory neurotransmitters in the regulation of pyloric relaxation and gastric emptying remains unclear. In this study, we investigated the effects of NO biosynthesis inhibitor, N G-nitro- l-arginine methyl ester ( l-NAME), and calcium dependent potassium channel blocker, apamin, on vagal stimulation-induced pyloric relaxation and gastric emptying in rats. Sodium nitroprusside (SNP), adenosine 5′-triphosphate (ATP), vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) caused pyloric relaxations in a dose dependent manner in vivo. Apamin (120 μg/kg) significantly reduced ATP and PACAP-induced pyloric relaxations without affecting SNP- or VIP-induced relaxations. Vagal stimulation (10 V, 1 ms, 1–20 Hz)-induced pyloric relaxation was significantly inhibited by l-NAME (10 mg/kg). The combined administration of l-NAME and apamin almost completely abolished vagal stimulation-induced pyloric relaxation. l-NAME and apamin significantly increased spontaneous contractions in the antrum, pylorus and duodenum. Increased motility index by l-NAME and apamin was significantly higher in the pylorus and duodenum, compared to that of antrum. l-NAME and apamin significantly delayed liquid gastric emptying. These results suggest that besides NO, probably ATP and PACAP, act as inhibitory neurotransmitters in the rat pylorus and regulate gastric emptying.