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Results for “Potassium Channel Blockers”

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PubMed2003

Iberiotoxin-induced block of Ca2+-activated K+ channels induces dihydropyridine sensitivity of ACh release from mammalian motor nerve terminals.

The role which Ca(2+)-activated K(+) (K(Ca)) channels play in regulating acetylcholine (ACh) release was examined at mouse motor nerve terminals. In particular, the ability of the antagonist iberiotoxin to recruit normally silent L-type Ca(2+) channels to participate in nerve-evoked release was examined using conventional intracellular electrophysiological techniques. Incubation of cut hemidiaphragm preparations with 10 microM nimodipine, a dihydropyridine L-type Ca(2+) channel antagonist, had no significant effect on quantal content of end-plate potentials. Nevertheless, 1 microM S-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K 8644) enhanced quantal content to 134.7 +/- 3.5% of control. Iberiotoxin (150 nM) increased quantal content to 177.5 +/- 9.9% of control, whereas iberiotoxin plus nimodipine increased quantal content to only 145.7 +/- 10.4% of control. Coapplication of 1 microM Bay K 8644 with iberiotoxin did not significantly increase quantal content further than did treatment with iberiotoxin alone. The effects of iberiotoxin and nimodipine alone or in combination on the miniature end-plate potential (MEPP) frequency following KCl-induced depolarization were examined using uncut hemidiaphragm preparations. Nimodipine alone had no effect on MEPP frequency from preparations incubated in physiological saline containing 5 to 20 mM KCl. Moreover, iberiotoxin alone or combined with nimodipine also had no effect on MEPP frequency in physiological salines containing 5 to 15 mM KCl. At 20 mM KCl, however, iberiotoxin significantly increased MEPP frequency to 125.6% of iberiotoxin-free values; combined treatment with nimodipine and iberiotoxin prevented this increase in MEPP frequency. Thus, loss of functional K(Ca) channels unmasks normally silent L-type Ca(2+) channels to participate in ACh release from motor nerve terminals, particularly under conditions of intense nerve terminal depolarization.

Acetylcholine↗

PubMed2000

Bistable behavior of inhibitory neurons controlling impulse traffic through the amygdala: role of a slowly deinactivating K+ current.

The intercalated cell masses of the amygdala are clusters of GABAergic neurons located strategically to influence behavioral responsiveness. Indeed, they receive glutamatergic sensory inputs from the basolateral amygdaloid complex and generate feedforward inhibition in neurons of the central amygdala that mediate important components of fear responses. In the present study, using whole-cell recording methods in coronal slices of the guinea pig amygdala, we show that the activity of intercalated neurons is a function of their recent firing history because they express an unusual voltage-dependent K(+) conductance (termed I(SD) for slowly deinactivating). This conductance activates in the subthreshold regime, inactivates in response to suprathreshold depolarizations, and deinactivates very slowly upon return to rest. As a result, after bouts of suprathreshold activity, these cells enter a self-sustaining state of heightened excitability associated with an increased input resistance and a membrane depolarization. In turn, these changes increase the likelihood that ongoing synaptic activity will trigger orthodromic action potentials. However, because each orthodromic spike "renews" the inactivation of I(SD), intercalated cells can remain hyperexcitable for a long time and, via the central amygdaloid nucleus, exert a lasting influence on behavior.

Action Potentials↗

PubMed1995

The involvement of ATP-sensitive potassium channels in beta-adrenoceptor-mediated vasorelaxation in the rat isolated mesenteric arterial bed.

1. We have used the isolated buffer-perfused superior mesenteric arterial bed of the rat to assess the involvement of ATP-sensitive potassium (KATP) channels in the vasorelaxant responses to beta-adrenoceptor agonists. 2. The vasorelaxant potencies of the non-selective beta-adrenoceptor agonist, isoprenaline, the beta 1-adrenoceptor agonist, dobutamine and the beta 2-adrenoceptor agonist, terbutaline were all significantly (P < 0.05) reduced (isoprenaline, ED50 = 265 +/- 31 pmol v. 1.05 +/- 0.42 nmol; dobutamine, ED50 = 294 +/- 67 pmol v. 497 +/- 115 pmol; terbutaline, ED50 = 157 +/- 26 nmol v. 452 +/- 120 nmol) in the presence of the KATP-channel blocker, glibenclamide. 3. The presence of glibenclamide only weakly influenced the vasorelaxant properties of salbutamol, a beta 2-adrenoceptor agonist, while those of verapamil, a beta-adrenoceptor-independent vasorelaxant, were unaffected. 4. In radioligand binding experiments, glibenclamide (1 nM-100 microM) did not displace any specific [3H]-dihydroalprenolol binding from rat beta-adrenoceptors. Therefore, glibenclamide does not bind to beta-adrenoceptors at the concentration used in the present investigation. 5. Vasorelaxant responses to dibutyryl cyclic AMP, the cell permeable analogue of cyclic AMP, were also unaffected by glibenclamide, indicating that the coupling of beta-adrenoceptors to KATP-channels occurs independently of the elevation of intracellular cyclic AMP. 6. We have shown that a significant element of the vasorelaxant responses to both beta 1- and beta 2-adrenoceptor activation involves the opening of KATP-channels. In conclusion, KATP-channels may play a physiological role in beta-adrenoceptor-mediated vasodilatation.

Adenosine Triphosphate↗

PubMed2003

Small conductance Ca2+-activated K+ channels formed by the expression of rat SK1 and SK2 genes in HEK 293 cells.

The rat SK1 gene (rSK1) does not form functional Ca2+-activated potassium channels when expressed alone in mammalian cell lines. Using a selective antibody to the rSK1 subunit and a yellow fluorescent protein (YFP) tag we have discovered that rSK1 expression produces protein that remains largely at intracellular locations. We tested the idea that rSK1 may need an expression partner, rSK2, in order to form functional channels. When rSK1 was co-expressed with rSK2 in HEK 293 cells it increased the current magnitude by 77 +/- 34% (as compared with cells expressing rSK2 alone). Co-expression of rSK1 with rSK2 also changed the channel pharmacology. The sensitivity of SK current to block by apamin was reduced approximately 16-fold from an IC50 of 94 pM (for SK2 alone) to 1.4 nM (for SK2 and SK1 together). The sensitivity to block by UCL 1848 (a potent small molecule blocker of SK channels) was similarly reduced, approximately 26-fold, from an IC50 of 110 pM to 2.9 nM. These data clearly demonstrate that rSK1 and rSK2 subunits interact. The most likely explanation for this is that the subunits are able to form heteromeric assemblies.

Algorithms↗

PubMed1999

Molecular basis, pharmacology and physiological role of cardiac K(ATP) channels.

ATP-dependent potassium (K(ATP)) channels exist in high density in the sarcolemmal membrane of heart muscle cells. Under normoxic conditions these channels are closed, but they become active when the intracellular ATP level falls. This leads to a shortening of the action potential duration, rendering the heart susceptible for life-threatening arrhythmias. Molecular biology has revealed that K(ATP) channels consist of heteromultimers of the inwardly rectifying channel Kir6.2 and the sulfonylurea receptor SUR. To date, three types of SURs were identified, representing the pancreatic (SUR1), the cardiac (SUR2A) and the smooth muscle (SUR2B) K(ATP) channel. In order to develop a novel therapeutic principle against ischemia-induced life-threatening arrhythmias leading to sudden cardiac death, the cardioselective K(ATP) channel blocker HMR 1883 was developed. This substance inhibits the sarcolemmal cardiac K(ATP) channel activated by the channel opener rilmakalim half-maximally at concentrations of 0.6-2.2 micromol/l, and substantially affects pancreatic K(ATP) channels at 9-50 times higher concentrations. K(ATP) channels of the coronary vascular system are only slightly blocked by HMR 1883 when activated by hypoxia. The substance was potently effective in preventing ventricular fibrillation in a conscious dog model, and thus can be considered to be a potential novel drug candidate against sudden cardiac death.

Adenosine Triphosphate↗

PubMed2003

Potassium channels in human umbilical artery cells.

OBJECTIVE: To identify K+ channels of smooth muscle of human umbilical artery using the patch-clamp technique and to study their effect on resting tone of umbilical artery rings. METHODS: Whole-cell and single-channel patch-clamp recordings in enzymatically isolated smooth muscle cells were made. Measurements of developed isometric force were performed on intact tissue. RESULTS: Delayed rectifier K+ channels (KDR) and large-conductance Ca2+-activated K+ channels (BKCa) contribute to the whole-cell voltage- and time-dependent outward K+ current, as it was specifically inhibited by 5 mM 4-aminopyridine (4-AP; KDR blocker) (92 +/- 4% at 0 mV, n = 7), by 1 mM tetraethylammonium (TEA; BKCa blocker) (71 +/- 4% at +60 mV, n = 4), and by 200 nM iberiotoxin (BKCa blocker) (64 +/- 7% at +60 mV, n = 4). In outside-out patches, BKCa channels had a single-channel conductance of 132 +/- 4 pS (n = 24) in asymmetric K+ conditions and 216 +/- 4 pS (n = 4) in a symmetric K+ gradient. The activity of the BKCa channels was significantly augmented by 1 microM Ca2+ in the inside-out configuration. 4-AP had no effect on resting tone of intact arterial rings. TEA produced contraction of arterial rings whereas phloretin, an activator of BKCa, relaxed them, which means that BKCa channels are functional in intact tissue and are involved in the maintenance of resting tone in this human vessel. CONCLUSION: The identities of K+ channels in the human umbilical artery were shown using the patch-clamp technique, and the physiologic effect of K+ channels on resting tone was documented.

4-Aminopyridine↗

PubMed1997

Stable expression and characterization of the human brain potassium channel Kv2.1: blockade by antipsychotic agents.

We have cloned the cDNA encoding the voltage-dependent K+ channel Kv2.1 from human brain (hKv2.1). RNase protection and RT-PCR (reverse transcriptase-PCR) experiments reveal abundant Kv2.1 transcripts in human brain with virtually no expression detectable in human heart. hKv2.1 has been stably transfected into a human glioblastoma cell line, and transformed cells display large, slowly activating outward currents. The kinetics, steady-state activation and inactivation parameters, and external tetraethylammonium sensitivity were all similar to those described previously for hKv2.1 channels transiently expressed in Xenopus oocytes or other mammalian cell lines. A number of dopamine receptor antagonist/antipsychotic agents were shown to block hKv2.1. Trifluoperizine, trifluperidol and pimozide produced time-dependent blockade of hKv2.1 with IC50 values of approx. 1-2 microM. The diphenylbutylpiperidine fluspirilene was shown to be 4-5-fold more potent than the other agents tested inhibiting hKv2.1 current with an IC50 value of 297 nM. The block produced by fluspirilene was both time- and frequency-dependent. Furthermore, fluspirilene (1 microM) shifted the midpotential of the hKv2.1 steady-state inactivation curve by approx. 15 mV in the hyperpolarizing direction. These results demonstrate the usefulness of this transfection system for the pharmacological characterization of hKv2. 1. Fluspirilene proved to be a relatively potent blocker of hKv2.1 and may provide a useful starting point for the development of more potent and selective agents active against this brain K+ channel.

Animals↗

PubMed2005

Blocking effect and crystal structure of natrin toxin, a cysteine-rich secretory protein from Naja atra venom that targets the BKCa channel.

Cysteine-rich secretory proteins (CRISPs) are widespread in snake venoms. Some members of these CRISPs recently have been found to block L-type Ca(2+) channels or cyclic nucleotide-gated ion (CNG) channels. Here, natrin purified from Naja atra venom, a member of the CRISP family, can induce a further contractile response in the endothelium-denuded thoracic aorta of mouse which has been contracted by a high-K(+) solution. Further experiments show it can block the high-conductance calcium-activated potassium (BK(Ca)) channel in a concentration-dependent manner with an IC(50) of 34.4 nM and a Hill coefficient of 1.02, which suggests that only a single natrin molecule is required to bind an ion channel to block BK(Ca) current. The crystal structure of natrin displaying two domains in tandem shows its cysteine-rich domain (CRD) has relatively independent flexibility, especially for the C-terminal long loop (loop I) of CRD to participate in the interface of two domains. On the basis of previous studies of CNG channel and L-Ca(2+) channel blockers, and the sequence and structural comparison of natrin and stecrisp, the deviation of the vital loop I of CRD is suggested to contribute to different effects of some CRISPs in protein-protein interaction.

Amino Acid Sequence↗

PubMed1995

Propofol-associated dilation of rat distal coronary arteries is mediated by multiple substances, including endothelium-derived nitric oxide.

Previous in vitro studies on the effect of propofol on coronary arteries have shown variable results, ranging from constriction to no effect to dilation. Although most of these studies reported that the observed effect is endothelium-independent, propofol also releases nitric oxide from cultured porcine endothelial cells. The present study examines the direct effect of propofol in rat distal coronary arteries in vitro, especially in regard to endothelial dependence and involvement of the adenosine triphosphate (ATP)-sensitive potassium channels (KATP channels). Forty-three subepicardial arteries (size 91.1 +/- 15.8 microns) from Wistar rats were studied in vitro in a no-flow, pressurized (40 mm Hg) state, using an optical density video detection system. After preconstriction with the thromboxane analog U46619 1 microM, relaxation responses to increasing concentrations of propofol (10(-6)-10(-4) M) were measured after 1) endothelial denudation, 2) pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine (L-NNA), 3) pretreatment with the cyclooxygenase inhibitor indomethacin, 4) pretreatment with the KATP channel blocker glibenclamide, or 5) no intervention (control). Propofol produced a significant concentration-dependent vasodilation of the U46619-preconstricted coronary arteries. This effect was significantly attenuated by endothelial denudation, pretreatment with L-NNA, or indomethacin, but was not affected by glibenclamide. We conclude that propofol has a direct vasodilatory effect on distal coronary arteries in rats. This effect is primarily endothelium-dependent and is mediated by multiple substances, including nitric oxide (NO) and a vasodilatory prostanoid. The effect is not mediated by opening of the K(ATP) channels.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMedDate not supplied

Sodium nitroprusside-induced rat fundus relaxation is ryanodine-sensitive and involves L-type Ca2+ channel and small conductance Ca(2+)-sensitive K+ channel components.

1 The aim of this study was to examine whether sodium nitroprusside (SNP)-induced relaxation of rat fundus longitudinal smooth muscle involves ryanodine-sensitive Ca2+ release. 2 SNP (300 nM-30 microM) elicited concentration-dependent relaxation of precontracted (1 microM carbachol) rat fundus, an effect almost abolished by the selective guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 microM). 3 SNP-mediated relaxations were almost abolished by 10 microM ryanodine. 4 SNP-mediated relaxations were also reduced by either 1 microM apamin (a selective small conductance Ca(2+)-sensitive K+ channel, SKCa, inhibitor) or the selective L-type Ca2+ channel inhibitor, nicardipine (3 microM). 5 SNP-induced relaxations were insensitive to 1 mM tetraethylammonium chloride (an inhibitor of large-conductance Ca(2+)-sensitive K+ channels) and 1 microM glibenclamide (an ATP-sensitive K+ channel inhibitor). 6 These data suggest that SNP-mediated fundus relaxation occurs via a cGMP-mediated and ryanodine-sensitive mechanism which requires, at least in part, SKCa and L-type Ca2+ channel activity.

Animals↗

PubMed2004

Mechanism of rectification in inward-rectifier K+ channels.

Inward rectifiers are a class of K+ channels that can conduct much larger inward currents at membrane voltages negative to the K+ equilibrium potential than outward currents at voltages positive to it, even when K+ concentrations on both sides of the membrane are made equal. This conduction property, called inward rectification, enables inward rectifiers to perform many important physiological tasks. Rectification is not an inherent property of the channel protein itself, but reflects strong voltage dependence of channel block by intracellular cations such as Mg2+ and polyamines. This voltage dependence results primarily from the movement of K+ ions across the transmembrane electric field along the pore, which is energetically coupled to the blocker binding and unbinding. This mutual displacement mechanism between several K+ ions and a blocker explains the signature feature of inward rectifier K+ channels, namely, that at a given concentration of intracellular K+, their macroscopic conductance depends on the difference between membrane voltage and the K+ equilibrium potential rather than on membrane voltage itself.

Animals↗

PubMed2006

Surface structure and its dynamic rearrangements of the KcsA potassium channel upon gating and tetrabutylammonium blocking.

KcsA is the first potassium channel for which the molecular structure was revealed. However, the high resolution structural information is limited to the transmembrane domain, and the dynamic picture of the full KcsA channel remains unsolved. We have developed a new approach to investigate the surface structure of proteins, and we applied this method to investigate the full length of the KcsA channel. Single-cysteine substitution was introduced into 25 sites, and specific reaction of these mutated channels to a bare surface of a flat gold plate was evaluated by surface plasmon resonance measurements. The surface plasmon resonance signals revealed the highest exposure for the mutant of the C-terminal end. When the gate of the KcsA channel is kept closed at pH 7.5, the extent of exposure showed periodic patterns for the consecutive sites located in the cytoplasmic (CP) and N-terminal domain. This suggests that these stretches take the alpha-helical structure. When the channel was actively gated at pH 4.0, many sites in the CP domain became exposed. Compared with the rigid structure in pH 7.5, these results indicate that the CP domain became loosely packed upon active gating. The C-terminal end of the M2 helix is a moving part of the gate, and it is exposed to the outer surface slightly at pH 4.0. By adding a channel blocker, tetrabutylammonium, the gate is further exposed. This suggests that in the active gating tetrabutylammonium keeps the gate open rather than being trapped in the central cavity.

Bacterial Proteins↗

PubMed2004

Spinal G-protein-gated K+ channels formed by GIRK1 and GIRK2 subunits modulate thermal nociception and contribute to morphine analgesia.

G-protein-gated potassium (K+) channels are found throughout the CNS in which they contribute to the inhibitory effects of neurotransmitters and drugs of abuse. Recent studies have implicated G-protein-gated K+ channels in thermal nociception and the analgesic action of morphine and other agents. Because nociception is subject to complex spinal and supraspinal modulation, however, the relevant locations of G-protein-gated K+ channels are unknown. In this study, we sought to clarify the expression pattern and subunit composition of G-protein-gated K+ channels in the spinal cord and to assess directly their contribution to thermal nociception and morphine analgesia. We detected GIRK1 (G-protein-gated inwardly rectifying K+ channel subunit 1) and GIRK2 subunits, but not GIRK3, in the superficial layers of the dorsal horn. Lack of either GIRK1 or GIRK2 was correlated with significantly lower expression of the other, suggesting that a functional and physical interaction occurs between these two subunits. Consistent with these findings, GIRK1 knock-out and GIRK2 knock-out mice exhibited hyperalgesia in the tail-flick test of thermal nociception. Furthermore, GIRK1 knock-out and GIRK2 knock-out mice displayed decreased analgesic responses after the spinal administration of higher morphine doses, whereas responses to lower morphine doses were preserved. Qualitatively similar data were obtained with wild-type mice after administration of the G-protein-gated K+ channel blocker tertiapin. We conclude that spinal G-protein-gated K+ channels consisting primarily of GIRK1/GIRK2 complexes modulate thermal nociception and mediate a significant component of the analgesia evoked by intrathecal administration of high morphine doses

Analgesia↗

PubMed2006

Early pharmacological preconditioning by erythropoietin mediated by inducible NOS and mitochondrial ATP-dependent potassium channels in the rat heart.

Administration of recombinant human erythropoietin (rhEPO) is known to induce protection against cardiac ischaemia injury improving functional recovery and reducing apoptosis. But the underlying mechanisms are not elucidated. We determined the role of nitric oxide synthases (NOS) as well as ATP-dependent (K(ATP)) and calcium-activated (K(Ca)) potassium channels in the early cardioprotection induced by rhEPO. Wistar male rats were divided into two experimental groups treated by rhEPO (5,000 IU/kg, i.p.) or saline (control group). One hour later, rats were anaesthetized, hearts isolated, retrogradely perfused and submitted to a 30-min no-flow global ischaemia followed by 120 min of reperfusion sequence. Cardiac functional recovery (left ventricular developed pressure, LVDP) was significantly higher in the group treated by rhEPO (LVDP at 30 min reperfusion: 71.7 +/- 2.3 mmHg) compared with the control group (57.4 +/- 5.8 mmHg). We observed the same significant effect on its derivative (dP/dt). The rhEPO-induced improvement in ventricular function was abolished by perfusion prior to ischaemia with either N-nitro-l-arginine methyl ester (l-NAME, a nonspecific NOS inhibitor) or N-(3-(aminomethyl)benzyl)acetamidine (1,400W, a specific inducible NOS inhibitor) or 5-hydroxydecanoic acid (5HD, a mitochondrial K(ATP) channel blocker) but not with paxilline (a K(Ca) channel inhibitor). Thus, in vivo rhEPO administration provides early preconditioning against ischaemic injury in the isolated perfused rat heart that is dependent on iNOS and mitochondrial K(ATP) channels.

Amidines↗

PubMed2001

Ca(2+) entry through L-type Ca(2+) channels helps terminate epileptiform activity by activation of a Ca(2+) dependent afterhyperpolarisation in hippocampal CA3.

In CA3 neurons of disinhibited hippocampal slice cultures the slow afterhyperpolarisation, following spontaneous epileptiform burst events, was confirmed to be Ca(2+) dependent and mediated by K(+) ions. Apamin, a selective blocker of the SK channels responsible for part of the slow afterhyperpolarisation reduced, but did not abolish, the amplitude of the post-burst afterhyperpolarisation. The result was an increased excitability of individual CA3 cells and the whole CA3 network, as measured by burst duration and burst frequency. Increases in excitability could also be achieved by strongly buffering intracellular Ca(2+) or by minimising Ca(2+) influx into the cell, specifically through L-type (but not N-type) voltage operated Ca(2+) channels. Notably the L-type Ca(2+) channel antagonist, nifedipine, was more effective than apamin at reducing the post-burst afterhyperpolarisation. Nifedipine also caused a greater increase in network excitability as determined from measurements of burst duration and frequency from whole cell and extracellular recordings. N-methyl D-aspartate receptor activation contributed to the depolarisations associated with the epileptiform activity but Ca(2+) entry via this route did not contribute to the activation of the post-burst afterhyperpolarisation. We suggest that Ca(2+) entry through L-type channels during an epileptiform event is selectively coupled to both apamin-sensitive and -insensitive Ca(2+) activated K(+) channels. Our findings have implications for how the route of Ca(2+) entry and subsequent Ca(2+) dynamics can influence network excitability during epileptiform discharges.

Animals↗

PubMed2001

Two distinct types of repetitive bursting activity mediated by NMDA in hypothalamic neurons in vitro.

Hypothalamic magnocellular dorsal nucleus neurons were recorded from adult guinea pig brain slices with the whole-cell patch-clamp technique to determine the effects of N-methyl-D-aspartate (NMDA) applied in the bath or by iontophoresis. In a majority of cells (59 of 77, 76.6%), rhythmic bursting discharges were evoked by specific activation of NMDA receptors when the membrane was more negative than -60 mV. This endogenous rhythmic activity was resistant to tetrodotoxin. It was suppressed by removal of extracellular Mg2+, indicating the involvement of the voltage-dependent block of the NMDA channel by Mg2+. Application of thapsigargin showed that rhythmic activity did not depend on the release of Ca2+ from reticulum stores. Blockers of Ca2+ conductances Ni2+ and nifedipine had no effects on the bursts. Their repolarization did not involve the activation of a strophantidin- or ouabain-sensitive pump, but partly depended on an apamine-sensitive Ca2+-dependent K+current. In a small subset of cells (9 of 69, 13%), specific activation of NMDA receptors induced another type of bursting activity which consisted of repetitive low-threshold spikes sustaining bursts of action potentials. Rhythmic low-threshold spikes subsisted in the presence of tetrodotoxin but were suppressed by Ni2+. Increasing the amount of NMDA brought about a switch from the rhythmic low-threshold spike burst firing to the rhythmic bursting activity observed for the majority of cells. The present data show for the first time that NMDA receptor activation can induce two independent rhythmic bursting behaviours in the same neuron, probably depending on the strength of the glutamatergic drive.

Action Potentials↗

PubMed2006

M1 muscarinic receptors inhibit L-type Ca2+ current and M-current by divergent signal transduction cascades.

Ion channels reside in a sea of phospholipids. During normal fluctuations in membrane potential and periods of modulation, lipids that directly associate with channel proteins influence gating by incompletely understood mechanisms. In one model, M(1)-muscarinic receptors (M(1)Rs) may inhibit both Ca(2+) (L- and N-) and K(+) (M-) currents by losing a putative interaction between channels and phosphatidylinositol-4,5-bisphosphate (PIP(2)). However, we found previously that M(1)R inhibition of N-current in superior cervical ganglion (SCG) neurons requires loss of PIP(2) and generation of a free fatty acid, probably arachidonic acid (AA) by phospholipase A(2) (PLA(2)). It is not known whether PLA(2) activity and AA also participate in L- and M-current modulation in SCG neurons. To test whether PLA(2) plays a similar role in M(1)R inhibition of L- and M-currents, we used several experimental approaches and found unanticipated divergent signaling. First, blocking resynthesis of PIP(2) minimized M-current recovery from inhibition, whereas L-current recovered normally. Second, L-current inhibition required group IVa PLA(2) [cytoplasmic PLA(2) (cPLA(2))], whereas M-current did not. Western blot and imaging studies confirmed acute activation of cPLA(2) by muscarinic stimulation. Third, in type IIa PLA(2) [secreted (sPLA(2))](-/-)/cPLA(2)(-/-) double-knock-out SCG neurons, muscarinic inhibition of L-current decreased. In contrast, M-current inhibition remained unaffected but recovery was impaired. Our results indicate that L-current is inhibited by a pathway previously shown to control M-current over-recovery after washout of muscarinic agonist. Our findings support a model of M(1)R-meditated channel modulation that broadens rather than restricts the roles of phospholipids and fatty acids in regulating ion channel activity.

Animals↗

PubMed2002

Position of aromatic residues in the S6 domain, not inactivation, dictates cisapride sensitivity of HERG and eag potassium channels.

Unintended block of HERG K+ channels is a side effect of many common medications and is the most common cause of acquired long QT syndrome associated with increased risk of life-threatening arrhythmias. The molecular mechanism of high-affinity HERG block by structurally diverse compounds has been attributed to pi-stacking and cation-pi interactions of a drug (e.g., cisapride) with specific aromatic amino acid residues (Tyr-652 and Phe-656) in the S6 alpha-helical domain that face the central cavity of the channel. It also has been proposed that strong C-type inactivation of HERG facilitates or is the primary determinant of high-affinity drug binding. The structurally related, but noninactivating eag channel is insensitive to HERG blockers unless inactivation is induced by specific amino acid mutations [Ficker, E., Jarolimek, W. & Brown, A. M. (2001) Mol. Pharmacol. 60, 1343-1348]. Here we examine the relative importance of inactivation vs. positioning of S6 aromatic residues in determining sensitivity of HERG and eag channels to block by cisapride. The repositioning of Tyr-652 or Phe-656 along the S6 alpha-helical domain of HERG reduced sensitivity of channels to block by cisapride. Moreover, independent of inactivation, repositioning of the equivalent aromatic residues in Drosophila eag channels induced sensitivity to block by cisapride. These findings suggest that positioning of S6 aromatic residues relative to the central cavity of the channel, not inactivation per se determines drug block of HERG or eag channels.

Amino Acid Sequence↗