Small-conductance Potassium Channels Research Articles

Role of endothelial large conductance Ca 2+ ‐activated potassium channels in blunted myogenic responsiveness of small skeletal muscle arteries after 48‐hour hypoxic exposure

Chronic hypoxia (CH) results in an endothelium-dependent blunting of myogenic responsiveness in isolated rat skeletal muscle arteries. We hypothesized that this attenuated myogenic responsiveness is due to an alteration of large conductance Ca2+-activated K+ (BKCa) channel activity in the endothelial cell of CH arteries. Third order branches of the caudal femoral artery (150–200 μm) from normoxic control rats and rats exposed to hypoxia (PB = 380 mmHg for 48 hours), were isolated, cannulated and pressurized. Vessel inner diameter was recorded at intraluminal pressures of 20–200 mmHg under normoxic conditions. Blunted myogenic responsiveness of CH arteries persisted in the presence of N-Nitro-L-arginine (L-NNA) (100 μM) and indomethacin (10 μM) and after further treatment with the luminally administered small and intermediate conductance potassium channel blockers, apamin (100 nM) and TRAM 34 (1 μM), respectively. However, myogenic responsiveness of CH arteries was restored to control levels by luminal administration of either a combination of charybdotoxin (100 nM) and apamin (100 nM) (n=5, both groups) or iberiotoxin (100 nM) alone (n=5, both groups), in the continued presence of L-NNA and indomethacin. These latter treatments did not affect myogenic reactivity in arteries from control rats. We conclude that endothelial cell BKCa channel activity is increased in CH arteries resulting in hyperpolarization that is conducted to the underlying vascular smooth muscle thereby attenuating myogenic responsiveness.

Intrinsic inhibitory innervation of the stomach

British Journal of Pharmacology (2005) 146, 163–164. doi:10.1038/sj.bjp.0706294

D-Tubocurarine-Sensitive Component of Calcium-Dependent Potassium Current in Guinea Pig Taenia Coli Myocytes

Using a patch-clamp technique in the whole-cell configuration, we studied transmembrane ion currents in isolated single smooth muscle cells of the guinea pig taenia coli. A depolarizing step shift of the membrane potential from −50 mV was accompanied by the appearance of an outward current. Application of d-tubocurarine (d-TK) or a nonselective blocker of voltage-dependent potassium channels, tetraethylammonium (TEA), led to a decrease in the outward current. Application of d-TK against the background of the action of TEA additionally decreased the outward current. Analysis of the current-voltage (I–V) relationships of the d-TK-sensitive current showed that this current is practically voltage-independent. At the same time, an inflection of the I–V curve of the potassium current within the segment of maximum activation of the voltage-dependent potassium current is indicative of the sensitivity of this current to the intracellular Ca2+ concentration. Therefore, the calcium-activated potassium current through small-conductance calcium-dependent potassium channels includes a d-TK-sensitive voltage-independent component. Using depolarizing shifts of the membrane potential, we observed high- and low-amplitude spontaneous outward currents (SOCs) in many studied cells, i.e., the effect of an increase in the conductance of calcium-dependent potassium channels as a result of periodic release of Ca2+ from the intracellular stores. Application of d-TK led to a decrease in the frequency of low-amplitude SOCs and exerted nearly no influence on the high-amplitude SOCs under study.

Two pathways for the activation of small-conductance potassium channels in neurons of substantia nigra pars reticulata

Neurons in substantia nigra pars reticulata express the messenger RNA for SK2 but not for SK3 subunits that form small-conductance, Ca 2+-dependent K + channels in dopamine neurons. To determine pathways for the activation of small-conductance, Ca 2+-dependent K + channels in substantia nigra pars reticulata neurons of rats and mice, we studied effects of the selective blocker of small-conductance, Ca 2+-dependent K + channels, apamin (0.01 or 0.3μM). Apamin diminished the afterhyperpolarization following each action potential and induced burst discharges in substantia nigra pars reticulata neurons. Apamin had a robust effect already at a low (10nM) concentration consistent with the expression of the SK2 subunit. Afterhyperpolarizations were also reduced by the Ca 2+ channel blockers Ni 2+ (100μM) and ω-conotoxin GVIA (1μM). Depletion of intracellular Ca 2+ stores did not change the afterhyperpolarization. However, we observed outward current pulses that occurred independently from action potentials and were abrogated by apamin. Apart from a faster time course, they shared all properties with spontaneous hyperpolarizations or outward currents that ryanodine receptor-mediated Ca 2+ release from intracellular stores induces in juvenile dopamine neurons. Sensitization of ryanodine receptors by caffeine silenced substantia nigra pars reticulata neurons. This effect was abolished by the depletion of intracellular Ca 2+ stores. We conclude that SK2 channels in substantia nigra pars reticulata neurons are activated by Ca 2+ influx through at least two types of Ca 2+ channels in the membrane and by ryanodine receptor-mediated Ca 2+ release from intracellular stores. Ryanodine receptors do not amplify small-conductance, Ca 2+-dependent K + channel activation by the Ca 2+ influx during a single spike. Yet, ryanodine receptor-mediated Ca 2+ release and, thereby, an activation of small-conductance, Ca 2+-dependent K + channels by intracellular Ca 2+ are available for excitability modulation in these output neurons of the basal ganglia system.

Adrenomedullin-induced endothelium-dependent relaxation in porcine ciliary arteries.

To investigate adrenomedullin-induced relaxation in isolated porcine ciliary arteries. In a myograph system (isometric force measurement), precontracted vessels ( approximately 0.1 micro M U46619; thromboxane A(2) analogue or approximately 10 nM endothelin-1) were exposed, in a cumulative manner, to increasing concentrations of adrenomedullin (1 nM to 1 micro M) in the presence or absence of different drugs. Some experiments were conducted in vessels with nonfunctional (intentionally mechanically damaged) endothelium. Adrenomedullin evoked marked relaxation [maximum relaxation (Rel(max)): 85.5 +/- 3.0%; negative log M concentration inducing 50% of Rel(max) (pD(2)): 7.4 +/- 0.1] in comparison to time-controls (Rel(max): 19.2 +/-4.8%; P < 0.001). Relaxation was inhibited by 3 micro M CGRP[8-37] (CGRP(1) receptor antagonist; Rel(max): 27.2% +/- 5.3%; P < 0.001) but not by 3 micro M adrenomedullin[22-52] (presumed adrenomedullin receptor antagonist; P = 0.75). Adrenomedullin-induced relaxation was less pronounced in nonfunctional endothelium vessels (Rel(max): 67.6% +/- 3.1%; pD(2): 6.9 +/- 0.1; P < 0.01). In vessels with functional endothelium, relaxation was not significantly influenced by 0.1 mM N(G)-nitro-L-arginine methyl ester (L-NAME; a nitric oxide synthesis inhibitor), 10 micro M indomethacin (a cyclooxygenase inhibitor), or 10 micro M 17-octadecynoic acid (a cytochrome P(450) inhibitor). In contrast, relaxation was significantly inhibited by either 10 mM tetraethylammonium (nonselective potassium channel inhibitor; P < 0.01) or 50 nM apamin (small conductance potassium channel inhibitor), together with 50 nM charybdotoxin (large and intermediate potassium channel inhibitor; P < 0.01). In the presence of these potassium channel inhibitors, the amount of relaxation was not significantly different (P > 0.50) from that observed in vessels with nonfunctional endothelium. In isolated porcine ciliary arteries, adrenomedullin induces relaxation that involves CGRP(1) receptors and is in part endothelium dependent. Endothelium-dependent relaxation was blocked by some potassium channel inhibitors, suggesting the possible release of an endothelium-derived hyperpolarizing factor (EDHF).

Extracellular ATP inhibits the small-conductance K channel on the apical membrane of the cortical collecting duct from mouse kidney.

We have used the patch-clamp technique to study the effects of changing extracellular ATP concentration on the activity of the small-conductance potassium channel (SK) on the apical membrane of the mouse cortical collecting duct. In cell-attached patches, the channel conductance and kinetics were similar to its rat homologue. Addition of ATP to the bathing solution of split-open single cortical collecting ducts inhibited SK activity. The inhibition of the channel by ATP was reversible, concentration dependent (K(i) = 64 microM), and could be completely prevented by pretreatment with suramin, a specific purinergic receptor (P(2)) blocker. Ranking of the inhibitory potency of several nucleotides showed strong inhibition by ATP, UTP, and ATP-gamma-S, whereas alpha, beta-Me ATP, and 2-Mes ATP failed to affect channel activity. This nucleotide sensitivity is consistent with P(2)Y(2) purinergic receptors mediating the inhibition of SK by ATP. Single channel analysis further demonstrated that the inhibitory effects of ATP could be elicited through activation of apical receptors. Moreover, the observation that fluoride mimicked the inhibitory action of ATP suggests the activation of G proteins during purinergic receptor stimulation. Channel inhibition by ATP was not affected by blocking phospholipase C and protein kinase C. However, whereas cAMP prevented channel blocking by ATP, blocking protein kinase A failed to abolish the inhibitory effects of ATP. The reduction of K channel activity by ATP could be prevented by okadaic acid, an inhibitor of protein phosphatases, and KT5823, an agent that blocks protein kinase G. Moreover, the effect of ATP was mimicked by cGMP and blocked by L-NAME (N(G)-nitro-l-arginine methyl ester). We conclude that the inhibitory effect of ATP on the apical K channel is mediated by stimulation of P(2)Y(2) receptors and results from increasing dephosphorylation by enhancing PKG-sensitive phosphatase activity.

Cloning and functional expression of a human liver CA 2+-sensitive, small-conductance potassium channel

Cloning and functional expression of a human liver CA 2+-sensitive, small-conductance potassium channel

Ca 2+-sensitive, small-conductance potassium channels contribute to cell volume recovery in human cholangiocarcinoma cells

Ca 2+-sensitive, small-conductance potassium channels contribute to cell volume recovery in human cholangiocarcinoma cells

Modulation by protease-activated receptors of the rat duodenal motility in vitro: possible mechanisms underlying the evoked contraction and relaxation.

1 The present study examined effects of agonist enzymes and receptor-activating peptides for protease-activated receptors (PARs) on duodenal motility in the rat, and also investigated possible mechanisms underlying the evoked responses. 2 Thrombin at 0.03-0.1 microM and the PAR-1-activating peptide SFLLR-NH2 at 3-100 microM or TFLLR-NH2 at 10-50 microM produced a dual action, relaxation followed by contraction of the duodenal longitudinal muscle. The PAR-2-activating peptide SLIGRL-NH2 at 10-100 microM elicited only small contraction. Trypsin at 0.08 microM induced small contraction, or relaxation followed by contraction, depending on preparations. The PAR-4-activating peptide GYPGKF-NH2 at 1000 microM exhibited no effect. 3 The contractile responses of the duodenal strips to TFLLR-NH2 and to SLIGRL-NH2 were partially attenuated by the L-type calcium channel blocker nifedipine (1 microM), the protein kinase C inhibitor GF109203X (1 microM) and the tyrosine kinase inhibitor genistein (15 microM), but were resistant to indomethacin (3 microM) and tetrodotoxin (1-10 microM). 4 The relaxation of the preparations exerted by TFLLR-NH2 was unaffected by indomethacin (3 microM), propranolol (5 microM), NG-nitro-L-arginine methyl ester (100 microM) and tetrodotoxin (1-10 microM). This relaxation was resistant to either GF109203X (1 microM) or genistein (15 microM), but was, remarkably, attenuated by combined application of these two kinase inhibitors. 5 Apamin (0.1 microM), an inhibitor of calcium-activated, small-conductance potassium channels, but not charybdotoxin (0.1 microM), completely abolished the PAR-1-mediated duodenal relaxation, and significantly enhanced the PAR-1-mediated contraction. 6 These findings demonstrate that PAR-1 plays a dual role, suppression and facilitation of smooth muscle motility in the rat duodenum, while PAR-2 plays a minor excitatory role in the muscle, and that PAR-4 is not involved in the duodenal tension modulation. The results also suggest that the contractile responses to PAR-1 and PAR-2 activation are mediated, in part, by activation of L-type calcium channels, protein kinase C and tyrosine kinase, and that the relaxation response to PAR-1 activation occurs via activation of apamin-sensitive, but charybdotoxin-insensitive, potassium channels, in which both protein kinase C and tyrosine kinase might be involved synergistically.

Small Conductance Potassium Channels Cause an Activity-Dependent Spike Frequency Adaptation and Make the Transfer Function of Neurons Logarithmic

We made a computational model of a single neuron to study the effect of the small conductance (SK) Ca 2+-dependent K + channel on spike frequency adaptation. The model neuron comprised a Na + conductance, a Ca 2+ conductance, and two Ca 2+-independent K + conductances, as well as a small and a large (BK) Ca 2+-activated K + conductance, a Ca 2+ pump, and mechanisms for Ca 2+ buffering and diffusion. Sustained current injection that simulated synaptic input resulted in a train of action potentials (APs) which in the absence of the SK conductance showed very little adaptation with time. The transfer function of the neuron was nearly linear, i.e., both asymptotic spike rate as well as the intracellular free Ca 2+ concentration ([Ca 2+] i) were approximately linear functions of the input current. Adding an SK conductance with a steep nonlinear dependence on [Ca 2+] i (Leinders and Vijverberg, 1992. Pflügers Arch. 422:223–232; Köhler, Hirschberg, Bond, Kinzie, Marrion, Maylie, and Adelman. 1996. Science. 273:1709–1714) caused a marked time-dependent spike frequency adaptation and changed the transfer function of the neuron from linear to logarithmic. Moreover, the input range the neuron responded to with regular spiking increased by a factor of 2.2. These results can be explained by a shunt of the cell resistance caused by the activation of the SK conductance. It might turn out that the logarithmic relationships between the stimuli of some modalities (e.g., sound or light) and the perception of the stimulus intensity (Fechner's law) have a cellular basis in the involvement of SK conductances in the processing of these stimuli.

Cellular localization and regulation of CHIF in kidney and colon.

Channel inducing factor (CHIF) is a novel cDNA recently cloned from a rat distal colon cDNA library of dexamethasone-treated animals. While its expression in Xenopus oocytes evokes a potassium channel activity similar to that induced by Isk (minK), its cellular role is not clear. CHIF exhibits significant homologies with proteins that are putatively regulatory (phospholemman, gamma-subunit of Na(+)-K(+)-ATPase, Mat-8) while it differs from the small-conductance potassium channel Isk. We have studied the tissue specificity of CHIF expression in rat by in situ hybridization. CHIF is selectively present in the distal parts of the nephron (medullary and papillary collecting ducts and end portions of cortical collecting tubule) and in the epithelial cells of the distal colon. No expression of CHIF was found in renal proximal tubule, loop of Henle and distal tubule, proximal colon, small intestine, lung, choroid plexus, salivary glands, or brain. To gain some insight into CHIF function, we have investigated, using in situ hybridization and ribonuclease protection assay, whether CHIF mRNA expression could be altered in some situations. In the distal colon, corticosteroid hormones, sodium restriction, low-potassium diet, and metabolic acidosis significantly increased CHIF mRNA expression. In the kidney, metabolic acidosis was the only condition that showed an increase in CHIF mRNA expression. Some of these treatments also altered the expression of the colonic H(+)-K(+)-ATPase mRNA. In summary, CHIF mRNA is selectively expressed in the medullary collecting duct of the kidney and in the epithelium of the distal colon; its expression varies differently in these two target tissues after alterations in corticosteroid status, potassium depletion, and metabolic acidosis. The precise cell-specific functions of CHIF remain to be established.

Pharmacology of the high-affinity apamin receptor in rabbit heart

Apamin is a potent blocker of calcium-activated small conductance potassium (SK) channels in neurons, liver, skeletal muscle and ileum smooth muscle, but not in cardiac muscle. Cardiac muscle is devoid of SK channels; however, in isolated, single ventricular myocytes apamin is an extremely potent blocker of the L-type calcium current, and the anti-arrhythmic drug quinidine reverses apamin block. To characterize the receptor binding properties and pharmacology of the apamin receptor in heart. The binding properties of the apamin receptor were determined by rapid filtration of purified rabbit heart membranes. Monoiodinated apamin binds to a labile, membrane-bound protein in heart membranes at a single, high-affinity site (KD = 8.07 +/- 2.14 pM and Bmax = 686 +/- 167 fmoles/mg protein, significant run test at P = 0.05 for a one site fit). 125I-apamin binding is dose-dependently inhibited by apamin, scyllatoxin, quinidine, amiloride, as well as a variety of di- and trivalent cations that are classical blockers of L-type calcium channels (e.g. Co2+, Cd2+, Mn2+, La3+, Gd3+). The cardiac apamin receptor is also critically dependent upon pH, temperature and KCl, and co-purifies in the same membrane fraction as L-type cardiac Ca2+ channels. The apamin receptor in rabbit heart P2 membranes has pharmacological and biochemical properties in common with both an SK channel and an L-type Ca2+ channel.

Cyclic AMP-dependent protein kinase activates small conductance potassium channels in liver cells

Cyclic AMP-dependent protein kinase activates small conductance potassium channels in liver cells

Dual effect of adenosine triphosphate on the apical small conductance K+ channel of the rat cortical collecting duct.

We used the patch-clamp technique to study the effects of ATP on the small-conductance potassium channel in the apical membrane of rat cortical collecting duct (CCD). This channel has a high open probability (0.96) in the cell-attached mode but activity frequently disappeared progressively within 1-10 min after channel excision (channel "run-down"). Two effects of ATP were observed. Using inside-out patches, low concentrations of ATP (0.05-0.1 mM) restored channel activity in the presence of cAMP-dependent protein kinase A (PKA). In contrast, high concentrations (1 mM) of adenosine triphosphate (ATP) reduced the open probability (Po) of the channel in inside-out patches from 0.96 to 0. 1.2 mM adenosine diphosphate (ADP) also blocked channel activity completely, but 2 mM adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP), a nonhydrolyzable ATP analogue, reduced Po only from 0.96 to 0.87. The half-maximal inhibition (Ki) of ATP and ADP was 0.5 and 0.6 mM, respectively, and the Hill coefficient of both ATP and ADP was close to 3. Addition of 0.2 or 0.4 mM ADP shifted the Ki of ATP to 1.0 and 2.0 mM, respectively. ADP did not alter the Hill coefficient. Reduction of the bath pH from 7.4 to 7.2 reduced the Ki of ATP to 0.3 mM. In contrast, a decrease of the free Mg2+ concentration from 1.6 mM to 20 microM increased the Ki of ATP to 1.6 mM without changing the Hill coefficient; ADP was still able to relieve the ATP-induced inhibition of channel activity over this low range of free Mg2+ concentrations. The blocking effect of ATP on channel activity in inside-out patches could be attenuated by adding exogenous PKA catalytic subunit to the bath. The dual effects of ATP on the potassium channel can be explained by assuming that (a) ATP is a substrate for PKA that phosphorylates the potassium channel to maintain normal function. (b) High concentrations of ATP inhibit the channel activity; we propose that the ATP-induced blockade results from inhibition of PKA-induced channel phosphorylation.