Hypotonicity-induced TRPV4 function in renal collecting duct cells: modulation by progressive cross-talk with Ca2+-activated K+ channels

The mouse cortical collecting duct (CCD) M-1 cells were grown to confluency on coverslips to assess the interaction between TRPV4 and Ca(2+)-activated K(+) channels. Immunocytochemistry demonstrated strong expression of TRPV4, along with the CCD marker, aquaporin-2, and the Ca(2+)-activated K(+) channels, the small conductance SK3 (K(Ca)2.3) channel and large conductance BKα channel (K(Ca)1.1). TRPV4 overexpression studies demonstrated little physical dependency of the K(+) channels on TRPV4. However, activation of TRPV4 by hypotonic swelling (or GSK1016790A, a selective agonist) or inhibition by the selective antagonist, HC-067047, demonstrated a strong dependency of SK3 and BK-α activation on TRPV4-mediated Ca(2+) influx. Selective inhibition of BK-α channel (Iberiotoxin) or SK3 channel (apamin), thereby depolarizing the cells, further revealed a significant dependency of TRPV4-mediated Ca(2+) influx on activation of both K(+) channels. It is concluded that a synergistic cross-talk exists between the TRPV4 channel and SK3 and BK-α channels to provide a tight functional regulation between the channel groups. This cross-talk may be progressive in nature where the initial TRPV4-mediated Ca(2+) influx would first activate the highly Ca(2+)-sensitive SK3 channel which, in turn, would lead to enhanced Ca(2+) influx and activation of the less Ca(2+)-sensitive BK channel.     

Ca2+-activated K+ channels in murine endothelial cells: block by intracellular calcium and magnesium

The intermediate (IK(Ca)) and small (SK(Ca)) conductance Ca(2+)-sensitive K(+) channels in endothelial cells (ECs) modulate vascular diameter through regulation of EC membrane potential. However, contribution of IK(Ca) and SK(Ca) channels to membrane current and potential in native endothelial cells remains unclear. In freshly isolated endothelial cells from mouse aorta dialyzed with 3 microM free [Ca(2+)](i) and 1 mM free [Mg(2+)](i), membrane currents reversed at the potassium equilibrium potential and exhibited an inward rectification at positive membrane potentials. Blockers of large-conductance, Ca(2+)-sensitive potassium (BK(Ca)) and strong inward rectifier potassium (K(ir)) channels did not affect the membrane current. However, blockers of IK(Ca) channels, charybdotoxin (ChTX), and of SK(Ca) channels, apamin (Ap), significantly reduced the whole-cell current. Although IK(Ca) and SK(Ca) channels are intrinsically voltage independent, ChTX- and Ap-sensitive currents decreased steeply with membrane potential depolarization. Removal of intracellular Mg(2+) significantly increased these currents. Moreover, concomitant reduction of the [Ca(2+)](i) to 1 microM caused an additional increase in ChTX- and Ap-sensitive currents so that the currents exhibited theoretical outward rectification. Block of IK(Ca) and SK(Ca) channels caused a significant endothelial membrane potential depolarization (approximately 11 mV) and decrease in [Ca(2+)](i) in mesenteric arteries in the absence of an agonist. These results indicate that [Ca(2+)](i) can both activate and block IK(Ca) and SK(Ca) channels in endothelial cells, and that these channels regulate the resting membrane potential and intracellular calcium in native endothelium.     

Bis-tetrahydroisoquinoline derivatives: AG525E1, a new step in the search for non-quaternary non-peptidic small conductance Ca(2+)-activated K(+) channel blockers

So far, small conductance Ca(2+)-activated K(+) channel (SK) blockers mostly consist of quaternary ammonium derivatives or peptides. Due to their physicochemical properties, these blockers are not suitable to study the physiological roles of SK channels in the central nervous system in vivo. Herein, we report the discovery of a chiral bis-tertiary amine with SK blocking properties from chemical modulation of laudanosine. AG525E1 has an affinity for SK channels (K(i)=293nM) approximately 100-fold higher than the tertiary compound laudanosine (K(i) approximately 30muM) and similar to the charged compound dequalinium (K(i)=221nM). AG525E1 equipotently blocks SK1, SK2 and SK3 currents in transfected cell lines. Because of its basic and lipophilic properties, it can reach central SK targets.     

Cloning and functional expression of a liver isoform of the small conductance Ca2+-activated K+ channel SK3

Small conductance Ca2+-activated K+ (SK) channels have been cloned from mammalian brain, but little is known about the molecular characteristics of SK channels in nonexcitable tissues. Here, we report the isolation from rat liver of an isoform of SK3. The sequence of the rat liver isoform differs from rat brain SK3 in five amino acid residues in the NH3 terminus, where it more closely resembles human brain SK3. SK3 immunoreactivity was detectable in hepatocytes in rat liver and in HTC rat hepatoma cells. Human embryonic kidney (HEK-293) cells transfected with liver SK3 expressed 10 pS K+ channels that were Ca2+ dependent (EC(50) 630 nM) and were blocked by the SK channel inhibitor apamin (IC(50) 0.6 nM); whole cell SK3 currents inactivated at membrane potentials more positive than -40 mV. Notably, the Ca2+ dependence, apamin sensitivity, and voltage-dependent inactivation of SK3 are strikingly similar to the properties of hepatocellular and biliary epithelial SK channels evoked by metabolic stress. These observations raise the possibility that SK3 channels influence membrane K+ permeability in hepatobiliary cells during liver injury.     

An amino acid outside the pore region influences apamin sensitivity in small conductance Ca2+-activated K+ channels

Small conductance calcium-activated potassium channels (SK, K(Ca)) are a family of voltage-independent K+ channels with a distinct physiology and pharmacology. The bee venom toxin apamin inhibits exclusively the three cloned SK channel subtypes (SK1, SK2, and SK3) with different affinity, highest for SK2, lowest for SK1, and intermediate for SK3 channels. The high selectivity of apamin made it a valuable tool to study the molecular makeup and function of native SK channels. Three amino acids located in the outer vestibule of the pore are of particular importance for the different apamin sensitivities of SK channels. Chimeric SK1 channels, enabling the homomeric expression of the rat SK1 (rSK1) subunit and containing the core domain (S1-S6) of rSK1, are apamin-insensitive. By contrast, channels formed by the human orthologue human SK1 (hSK1) are sensitive to apamin. This finding hinted at the involvement of regions beyond the pore as determinants of apamin sensitivity, because hSK1 and rSK1 have an identical amino acid sequence in the pore region. Here we investigated which parts of the channels outside the pore region are important for apamin sensitivity by constructing chimeras between apamin-insensitive and -sensitive SK channel subunits and by introducing point mutations. We demonstrate that a single amino acid situated in the extracellular loop between the transmembrane segments S3 and S4 has a major impact on apamin sensitivity. Our findings enabled us to convert the hSK1 channel into a channel that was as sensitive for apamin as SK2, the SK channel with the highest sensitivity.     

Modulation of endothelial SK3 channel activity by Ca²⁺-dependent caveolar trafficking

Small- and intermediate-conductance Ca(2+)-activated K(+) channels (SK3/Kcnn3 and IK1/Kcnn4) are expressed in vascular endothelium. Their activities play important roles in regulating vascular tone through their modulation of intracellular concentration ([Ca(2+)](i)) required for the production of endothelium-derived vasoactive agents. Activation of endothelial IK1 or SK3 channels hyperpolarizes endothelial cell membrane potential, increases Ca(2+) influx, and leads to the release of vasoactive factors, thereby impacting blood pressure. To examine the distinct roles of IK1 and SK3 channels, we used electrophysiological recordings to investigate IK1 and SK3 channel trafficking in acutely dissociated endothelial cells from mouse aorta. The results show that SK3 channels undergo Ca(2+)-dependent cycling between the plasma membrane and intracellular organelles; disrupting Ca(2+)-dependent endothelial caveolae cycling abolishes SK3 channel trafficking. Moreover, transmitter-induced changes in SK3 channel activity and surface expression modulate endothelial membrane potential. In contrast, IK1 channels do not undergo rapid trafficking and their activity remains unchanged when either exo- or endocytosis is block. Thus modulation of SK3 surface expression may play an important role in regulating endothelial membrane potential in a Ca(2+)-dependent manner.     

Role of potassium channels in catecholamine secretion in the rat adrenal gland

We elucidated the functional contribution of K(+) channels to cholinergic control of catecholamine secretion in the perfused rat adrenal gland. The small-conductance Ca(2+)-activated K(+) (SK(Ca))-channel blocker apamin (10-100 nM) enhanced the transmural electrical stimulation (ES; 1-10 Hz)- and 1, 1-dimethyl-4-phenyl-piperazinium (DMPP; 5-40 microM)-induced increases in norepinephrine (NE) output, whereas it did not affect the epinephrine (Epi) responses. Apamin enhanced the catecholamine responses induced by acetylcholine (6-200 microM) and methacholine (10-300 microM). The putative large-conductance Ca(2+)-activated K(+) channel blocker charybdotoxin (10-100 nM) enhanced the catecholamine responses induced by ES, but not the responses induced by cholinergic agonists. Neither the K(A) channel blocker mast cell degranulating peptide (100-1000 nM) nor the K(V) channel blocker margatoxin (10-100 nM) affected the catecholamine responses. These results suggest that SK(Ca) channels play an inhibitory role in adrenal catecholamine secretion mediated by muscarinic receptors and also in the nicotinic receptor-mediated secretion of NE, but not of Epi. Charybdotoxin-sensitive Ca(2+)-activated K(+) channels may control the secretion at the presynaptic site.     

IK channels are involved in the regulatory volume decrease in human epithelial cells

Parallel activation ofCa²⁺-dependent K⁺ channels and volume-sensitiveCl channels is known to be responsible for KCl effluxduring regulatory volume decrease (RVD) in human epithelial Intestine407 cells. The present study was performed to identify theK⁺ channel type. RT-PCR demonstrated mRNA expression ofCa²⁺-activated, intermediate conductance K⁺(IK), but not small conductance K⁺ (SK1) or largeconductance K⁺ (BK) channels in this cell line. Whole cellrecordings showed that ionomycin or hypotonic stress activated inwardlyrectifying K⁺ currents that were reversibly blocked by IKchannel blockers [clotrimazole (CLT) and charybdotoxin] but not by SKand BK channel blockers (apamin and iberiotoxin). Inside-out recordingsrevealed the existence of CLT-sensitive single K⁺-channelactivity, which exhibited an intermediate unitary conductance (30 pS at100 mV). The channel was activated by cytosolic Ca²⁺ ininside-out patches and by a hypotonic challenge in cell-attached patches. The RVD was suppressed by CLT, but not by apamin oriberiotoxin. Thus we conclude that the IK channel is involved in theRVD process in these human epithelial cells.

     

Essential role of EDHF in the initiation and maintenance of adrenergic vasomotion in rat mesenteric arteries

The possible roles of endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)), nitric oxide (NO), arachidonic acid (AA) metabolites, and Ca(2+)-activated K(+) (K(Ca)) channels in adrenergically induced vasomotion were examined in pressurized rat mesenteric arteries. Removal of the endothelium or buffering [Ca(2+)](i) selectively in endothelial cells with BAPTA eliminated vasomotion in response to phenylephrine (PE; 10.0 microM). In arteries with intact endothelium, inhibition of NO synthase with N(omega)-nitro-l-arginine methyl ester (l-NAME; 300.0 microM) or N(omega)-nitro-l-arginine (l-NNA; 300.0 microM) did not eliminate vasomotion. Neither inhibition of cGMP formation with 10.0 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) nor inhibition of prostanoid formation (10.0 microM indomethacin) eliminated vasomotion. Similarly, inhibition of AA cytochrome P-450 metabolism with an intraluminal application of 17-octadecynoic acid (17-ODYA) or 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) failed to eliminate vasomotion. In contrast, intraluminal application of the K(Ca) channel blockers apamin (250.0 nM) and charybdotoxin (100.0 nM), together, abolished vasomotion and changed synchronous Ca(2+) oscillations in smooth muscle cells to asynchronous propagating Ca(2+) waves. Apamin, charybdotoxin, or iberiotoxin (100.0 nM) alone did not eliminate vasomotion, nor did the combination of apamin and iberiotoxin. The results show that adrenergic vasomotion in rat mesenteric arteries is critically dependent on Ca(2+)-activated K(+) channels in endothelial cells. Because these channels (small- and intermediate-conductance K(Ca) channels) are a recognized component of EDHF, we conclude therefore that EDHF is essential for the development of adrenergically induced vasomotion.     

Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells

We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.     

Interaction of SK(Ca) channels and L-type Ca(2+) channels in catecholamine secretion in the rat adrenal gland

We elucidated the interaction of small-conductance Ca(2+)-activated K(+) (SK(Ca)) channels and L-type Ca(2+) channels in muscarinic receptor-mediated control of catecholamine secretion in the isolated perfused rat adrenal gland. The muscarinic agonist methacholine (10-300 microM) produced concentration-dependent increases in adrenal output of epinephrine and norepinephrine. The SK(Ca) channel blocker apamin (1 microM) enhanced the methacholine-induced catecholamine responses. The facilitatory effect of apamin on the methacholine-induced catecholamine responses was not observed during treatment with the L-type Ca(2+) channel blocker nifedipine (3 microM) or Ca(2+)-free solution. Nifedipine did not affect the methacholine-induced catecholamine responses, but it inhibited the responses during treatment with apamin. The L-type Ca(2+) channel activator Bay k 8644 (1 microM) enhanced the methacholine-induced catecholamine responses, whereas the enhancement of the methacholine-induced epinephrine and norepinephrine responses were prevented and attenuated by apamin, respectively. These results suggest that SK(Ca) channels are activated by muscarinic receptor stimulation, which inhibits the opening of L-type Ca(2+) channels and thereby attenuates adrenal catecholamine secretion.     

Role of small conductance calcium-activated potassium channels expressed in PVN in regulating sympathetic nerve activity and arterial blood pressure in rats

Small conductance Ca(2+)-activated K(+) (SK) channels regulate membrane properties of rostral ventrolateral medulla (RVLM) projecting hypothalamic paraventricular nucleus (PVN) neurons and inhibition of SK channels increases in vitro excitability. Here, we determined in vivo the role of PVN SK channels in regulating sympathetic nerve activity (SNA) and mean arterial pressure (MAP). In anesthetized rats, bilateral PVN microinjection of SK channel blocker with peptide apamin (0, 0.125, 1.25, 3.75, 12.5, and 25 pmol) increased splanchnic SNA (SSNA), renal SNA (RSNA), MAP, and heart rate (HR) in a dose-dependent manner. Maximum increases in SSNA, RSNA, MAP, and HR elicited by apamin (12.5 pmol, n = 7) were 330 ± 40% (P < 0.01), 271 ± 40% (P < 0.01), 29 ± 4 mmHg (P < 0.01), and 34 ± 9 beats/min (P < 0.01), respectively. PVN injection of the nonpeptide SK channel blocker UCL1684 (250 pmol, n = 7) significantly increased SSNA (P < 0.05), RSNA (P < 0.05), MAP (P < 0.05), and HR (P < 0.05). Neither apamin injected outside the PVN (12.5 pmol, n = 6) nor peripheral administration of the same dose of apamin (12.5 pmol, n = 5) evoked any significant changes in the recorded variables. PVN-injected SK channel enhancer 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO, 5 nmol, n = 4) or N-cyclohexyl-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidin]amine (CyPPA, 5 nmol, n = 6) did not significantly alter the SSNA, RSNA, MAP, and HR. Western blot and RT-PCR analysis of punched PVN tissue showed abundant expression of SK1-3 channels. We conclude that SK channels expressed in the PVN play an important role in the regulation of sympathetic outflow and cardiovascular function.     

Isosteviol acts on potassium channels to relax isolated aortic strips of Wistar rat

Isosteviol is a derivative of stevioside, a constituent of Stevia rebaudiana, which is commonly used as a noncaloric sugar substitute in Japan and Brazil. In the present study, the role of potassium channels in the vasodilator effect of isosteviol was investigated using potassium channel blockers on isosteviol-induced relaxation of isolated aortic rings prepared from Wistar rats. Isosteviol dose-dependently relaxed the vasopressin (10(-8) M)-induced vasoconstriction in isolated aortic rings with or without endothelium. However, in the presence of potassium chloride (3x10(-2) M), the vasodilator effect of isosteviol on arterial strips disappeared. Only the inhibitors specific for the ATP-sensitive potassium (K(ATP)) channel or small conductance calcium-activated potassium (SK(Ca)) channel inhibited the vasodilator effect of isosteviol in isolated aortic rings contracted with 10(-8) M vasopressin. Also; since the isosteviol-induced relaxation was unchanged by methylene blue, a role of nitric oxide and/or endothelium in the vasodilatation produced by isosteviol could be ruled out. The obtained results indicated that vasodilatation induced by isosteviol is related to the opening of SK(Ca) and K(ATP) channels.     

Tamapin,a venom peptide from the Indian red scorpion (Mesobuthus tamulus) that targets small conductance Ca2+-activated K+ channels and afterhyperpolarization currents in central neurons

The biophysical properties of small conductance Ca(2+)-activated K(+) (SK) channels are well suited to underlie afterhyperpolarizations (AHPs) shaping the firing patterns of a conspicuous number of central and peripheral neurons. We have identified a new scorpion toxin (tamapin) that binds to SK channels with high affinity and inhibits SK channel-mediated currents in pyramidal neurons of the hippocampus as well as in cell lines expressing distinct SK channel subunits. This toxin distinguished between the SK channels underlying the apamin-sensitive I(AHP) and the Ca(2+)-activated K(+) channels mediating the slow I(AHP) (sI(AHP)) in hippocampal neurons. Compared with related scorpion toxins, tamapin displayed a unique, remarkable selectivity for SK2 versus SK1 ( approximately 1750-fold) and SK3 ( approximately 70-fold) channels and is the most potent SK2 channel blocker characterized so far (IC(50) for SK2 channels = 24 pm). Tamapin will facilitate the characterization of the subunit composition of native SK channels and help determine their involvement in electrical and biochemical signaling.     

Small-conductance, Ca(2+) -activated K+ channel 2 is the key functional component of SK channels in mouse urinary bladder

Small-conductance Ca(2+)-activated K(+) (SK) channels play an important role in regulating the frequency and in shaping urinary bladder smooth muscle (UBSM) action potentials, thereby modulating contractility. Here we investigated a role for the SK2 member of the SK family (SK1-3) utilizing: 1) mice expressing beta-galactosidase (beta-gal) under the direction of the SK2 promoter (SK2 beta-gal mice) to localize SK2 expression and 2) mice lacking SK2 gene expression (SK2(-/-) mice) to assess SK2 function. In SK2 beta-gal mice, UBSM staining was observed, but staining was undetected in the urothelium. Consistent with this, urothelial SK2 mRNA was determined to be 4% of that in UBSM. Spontaneous phasic contractions in wild-type (SK2(+/+)) UBSM strips were potentiated (259% of control) by the selective SK channel blocker apamin (EC(50) = 0.16 nM), whereas phasic contractions of SK2(-/-) strips were unaffected. Nerve-mediated contractions of SK2(+/+) UBSM strips were also increased by apamin, an effect absent in SK2(-/-) strips. Apamin increased the sensitivity of SK2(+/+) UBSM strips to electrical field stimulation, since pretreatment with apamin decreased the frequency required to reach a 50% maximal contraction (vehicle, 21 +/- 4 Hz, n = 6; apamin, 12 +/- 2 Hz, n = 7; P < 0.05). In contrast, the sensitivity of SK2(-/-) UBSM strips was unaffected by apamin. Here we provide novel insight into the molecular basis of SK channels in the urinary bladder, demonstrating that the SK2 gene is expressed in the bladder and that it is essential for the ability of SK channels to regulate UBSM contractility.     

TWIK-related acid-sensitive K+ channel 1 (TASK1) and TASK3 critically influence T lymphocyte effector functions

Two major K(+) channels are expressed in T cells, (i) the voltage-dependent K(V)1.3 channel and (ii) the Ca(2+)-activated K(+) channel KCa 3.1 (IKCa channel). Both critically influence T cell effector functions in vitro and animal models in vivo. Here we identify and characterize TWIK-related acid-sensitive potassium channel 1 (TASK1) and TASK3 as an important third K(+) conductance on T lymphocytes. T lymphocytes constitutively express TASK1 and -3 protein. Application of semi-selective TASK blockers resulted in a significant reduction of cytokine production and cell proliferation. Interference with TASK channels on CD3(+) T cells revealed a dose-dependent reduction ( approximately 40%) of an outward current in patch clamp recordings indicative of TASK channels, a finding confirmed by computational modeling. In vivo relevance of our findings was addressed in an experimental model of multiple sclerosis, adoptive transfer experimental autoimmune encephalomyelitis. Pretreatment of myelin basic protein-specific encephalitogenic T lymphocytes with TASK modulators was associated with significant amelioration of the disease course in Lewis rats. These data introduce K(2)P channels as novel potassium conductance on T lymphocytes critically influencing T cell effector function and identify a possible molecular target for immunomodulation in T cell-mediated autoimmune disorders.     

Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels

This study examines the roles of voltage-dependent Ca(2+) channels (VDCC), ryanodine receptors (RyRs), large-conductance Ca(2+)-activated K(+) (BK) channels, and small-conductance Ca(2+)-activated K(+) (SK) channels in the regulation of phasic contractions of guinea pig urinary bladder smooth muscle (UBSM). Nisoldipine (100 nM), a dihydropyridine inhibitor of VDCC, abolished spontaneous UBSM contractions. Ryanodine (10 microM) increased contraction frequency and thereby integrated force and, in the presence of the SK blocker apamin, had a greater effect on integrated force than ryanodine alone. Blocking BK (iberiotoxin, 100 nM) or SK (apamin, 100 nM) channels increased contraction amplitude and duration but decreased frequency. The contractile response to iberiotoxin was more pronounced than to apamin. The increases in contraction amplitude and duration to apamin were substantially augmented with ryanodine pretreatment. These results indicate that BK and SK channels have prominent roles as negative feedback elements to limit UBSM contraction amplitude and duration. RyRs also appear to play a significant role as a negative feedback regulator of contraction frequency and duration, and this role is influenced by the activity of SK channels.