Regulation of Kir2.1 channels by the Rho-GTPase, Rac1.

Mutations in Kir2.1 inwardly rectifying potassium channels are associated with Andersen syndrome, a disease characterized by potentially fatal cardiac arrhythmias. While several Andersen-associated mutations affect membrane expression, the cytoplasmic signals that regulate Kir2.1 trafficking are poorly understood. Here, we investigated whether the Rho-family of small GTPases regulates trafficking of Kir2.1 channels expressed in HEK-293 cells. Treatment with Clostridium difficile toxin B, an inhibitor of Rho-family GTPases, or co-expression of the dominant-negative mutant of Rac1 (Rac1(DN)) increased Kir2.1 channels approximately 2-fold. However, the dominant-negative forms of other Rho-family GTPases, RhoA or Cdc42, did not alter Kir2.1 currents, suggesting a selective effect of Rac1 on Kir2.1 channels. Single-channel properties (gamma, tau(o), tau(c)) and total protein levels of Kir2.1 were unchanged with co-expression of Rac1(DN); however, studies using TIRF microscopy and CFP-tagged Kir2.1 revealed increased channel surface expression. Immunohistochemical detection of extracellularly tagged HA-Kir2.1 channels showed that Rac1(DN) reduced channel internalization when co-expressed. Finally, the dominant-negative mutant of dynamin, which interferes with endocytosis, occluded the Rac1(DN)-induced potentiation of Kir2.1 currents. These data suggest that inhibition of Rac1 increases Kir2.1 surface expression by interfering with endocytosis, likely via a dynamin-dependent pathway. Surprisingly, Rac1(DN) did not alter Kir2.2 current density or internalization, suggesting subunit specific modulation of Kir2.1 channels. Consistent with this, construction of Kir2.1/2.2 chimeras implicated the C-terminal domain of Kir2.1 in mediating the potentiating effect of Rac1(DN). This novel pathway for regulating surface expression of cardiac Kir2.1 channels could have implications for normal and diseased cardiac states.

Carbachol induces secretion in a mast cell line (RBL-2H3) transfected with the ml muscarinic receptor gene.

The possibility that the ml muscarinic receptor subtype can induce release of intracellular granules and transmitters was studied by transfecting a cultured mast cell line. RBL-2H3 cells, with the ml receptor gene. Comparisons were made between carbachol- and antigen-induced activation of various secretory responses. Like antigen, carbachol stimulated inositol phospholipid hydrolysis and release of arachidonic acid with concomitant dose-dependent secretion of granular contents. Carbachol also stimulated a biphasic increase in intracellular calcium, as measured by single cell fura-2 measurements. Although the kinetics of the carbachol-induced rise in intracellular calcium differed from that induced by antigen, they both utilized the same intracellular pool of calcium, and the second phase of the rise in intracellular calcium was dependent on extracellular calcium in both cases. Thus, the ml muscarinic receptor activates release of granules by a mechanism ostensibly similar to that of antigen.

Dual modulation of an inwardly rectifying potassium conductance.

The modulation of a constitutively active IRK1-like inwardly rectifying potassium channel, that is endogenously expressed in the RBL-2H3 cell, was studied with the whole-cell patch-clamp technique. Activation of G-proteins by intracellular application of GTP gamma S revealed a dual modulation of the inward rectifier. An initial increase in inward current amplitude was induced by GTP gamma S, followed by a profound inhibition of the current. The stimulation of the inward rectifier by GTP gamma S was abolished by pretreatment with pertussis toxin. The inhibitory phase of the GTP gamma S-induced response was pertussis toxin-insensitive. Stimulation of the m1-muscarinic receptor expressed in the RBL cell after stable transfection, induced an inhibition of the inwardly rectifying currents. Application of protein kinase C activators such as phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate, resulted in a strong inhibition of the currents. Application of the cAMP-dependent protein kinase activator 8-bromo cAMP also induced an inhibition of the inward rectifier. It is concluded that the inward rectifier of the RBL-2H3 cell may be inhibited both by activation of protein kinase C and by cAMP-dependent protein kinase. As this type of inward rectifier is widely expressed in the nervous system, these data imply that the channel can be inhibited by receptors that stimulate phospholipase C and/or stimulate adenylyl cyclase, and can be activated by receptors that inhibit adenylyl cyclase activity.

GTP-binding protein Gq mediates muscarinic-receptor-induced inhibition of the inwardly rectifying potassium channel IRK1 (Kir 2.1).

The inwardly rectifying potassium channel IRK1, a member of the Kir 2.0 family, is inhibited by m1 muscarinic receptor stimulation. In this study the mechanism of action underlying the muscarinic response was investigated by identification of the subtype of heterotrimeric G-protein involved in transduction of the signal. tsA201 cells were simultaneously transfected with cDNAs encoding IRK1, m1 and the Galpha subunit of either G(q), G(12) or G(13). The whole-cell patch-clamp technique was used to study the effects of G-protein transfection. Antibodies generated against the C-terminal regions of Galpha(q/11) and Galpha(12) were used to confirm G-protein expression by Western blot. When challenged with carbachol, IRK1 currents recorded from cells co-transfected with Galpha(q) were potently inhibited compared with controls. Conversely, co-transfection with Galpha(12) or Galpha(13) subunits had no effect on muscarinic-receptor-induced inhibition of IRK1. Concentration response curves revealed that carbachol was 16 times more potent at inhibiting IRK1 currents in cells co-transfected with Galpha(q) as compared with Galpha(12) co-transfected cells. Immunoblotting illustrated low levels of endogenous Galpha(q/11) and Galpha(12) in untransfected tsA cells. Transfection with Galpha(q) or Galpha(12) cDNAs greatly increased the levels of G-protein expression in both cell populations. G-protein expression did not interfere with m1 muscarinic receptor expression levels. These findings suggest that the m1 muscarinic-receptor-induced inhibition of IRK1 is mediated by the heterotrimeric G-protein, Galpha(q), in tsA cells.

A kinetic analysis of the endplate ion channel blocking action of disopyramide and its optical isomers.

The effects of the antiarrhythmic agent disopyramide was studied on responses from voltage-clamped endplates at the neuromuscular junction of the garter snake. Disopyramide reduced endplate current amplitude and decay time constant in a concentration- and voltage-dependent manner. Endplate current decays remained monophasic in the presence of the drug. These results were interpreted in terms of the drug blocking the open form of the acetylcholine receptor-ion channel complex. Disopyramide produced a greater reduction of the amplitude of endplate currents than of miniature endplate currents. The reduction in miniature endplate current amplitude was not voltage-dependent. Analysis of endplate current driving functions showed that this was due to the rapid occurrence of channel block during the rising phase of the endplate current. The residual reduction, apart from that produced by channel block, is most probably due to receptor block. Disopyramide had a voltage-dependent blocking rate constant of about 10(7) M-1 S-1 at -90 mV. The unblocking rate constant was estimated from the results of experiments using paired ionophoretically applied pulses of acetylcholine. This value was again voltage-dependent and approximately 1 s-1. The actions of the (+)- and (-)-stereoisomers of disopyramide on endplate current decay were identical, indicating that the channel binding site at the neuromuscular junction is not stereoselective.

Muscarinic acetylcholine receptor subtypes: localization and structure/function.

Based on the sequence of the five cloned muscarinic receptor subtypes (m1-m5), subtype selective antibody and cDNA probes have been prepared. Use of these probes has demonstrated that each of the five subtypes has a markedly distinct distribution within the brain and among peripheral tissues. The distributions of these subtypes and their potential physiological roles are discussed. By use of molecular genetic manipulation of cloned muscarinic receptor cDNAs, the regions of muscarinic receptors that specify G-protein coupling and ligand binding have been defined in several recent studies. Overall, these studies have shown that amino acids within the third cytoplasmic loop of the receptors define their selectivities for different G-proteins and that multiple discontinuous epitopes contribute to their selectivities for different ligands. The residues that contribute to ligand binding and G-protein coupling are described, as well as the implied structures of these functional domains.

Electrophysiological responses to muscarinic receptor stimulation in cultured hippocampal neurons.

Electrophysiological recordings of responses to muscarinic receptor stimulation in cultured embryonic hippocampal neurons have been largely unsuccessful to date. In this study muscarinic receptor binding was demonstrated in 2-week-old embryonic rat hippocampal cultures. Using whole-cell patch-clamp recording we found that 1-5 microM carbachol produced multiple effects including depolarization, increased action potential firing rate, increased synaptic activity and a reduction in the amplitude of medium-duration afterhyperpolarizations. Voltage-clamp analysis revealed a time-dependent current relaxation with hyperpolarizing steps from a holding potential of about -40 mV which was inhibited by 10 microM muscarine or 50 microM carbachol and had characteristics similar to those of the m-current. Both atropine and pirenzepine inhibited all of these effects indicating that these cholinergic actions were mediated by muscarinic receptors. This study shows that muscarinic responses obtained classically in hippocampal brain slices can also be produced in cultured hippocampus.

Exaggerated effects of progestogen on uterine artery pulsatility index in Turner's syndrome patients receiving hormone replacement therapy.

OBJECTIVE: To investigate the effect of estrogen and progestogen on the resistance to blood flow in the uterine arteries of Turner's syndrome patients. DESIGN: Prospective clinical study. SETTING: A tertiary infertility clinic. PATIENTS: Five Turner's syndrome patients, six patients who had surgical castration, and five patients with idiopathic primary ovarian failure. INTERVENTIONS: The patients were treated with 2 mg E2 valerate to which 500 micrograms norgesterel was added for 10 days in a 28-day cycle. Transvaginal color Doppler was used to measure pulsatility index in the uterine arteries at eight regular intervals during a single cycle. MAIN OUTCOME MEASURE: Pulsatility index of the uterine arteries. RESULTS: The administration of norgesterel to Turner's syndrome patients resulted in an increase in pulsatility index that was significantly higher than in patients who had surgical castration (confidence interval = 0.17 to 2.42). CONCLUSION: The uterine arteries of Turner's syndrome patients are more sensitive to the tonic effect of progestogen. If manifest in cardiac arteries also this phenomenon may be partly responsible for the increased incidence of cardiovascular disease and shorter life expectancy in Turner's syndrome patients. To achieve optimal protection from cardiovascular disease, Turner's syndrome patients may benefit from hormone replacement treatment containing altered doses of estrogen and progestogen.

m4 muscarinic receptor subtype activates an inwardly rectifying potassium conductance in AtT20 cells.

The modulation of an inwardly rectifying potassium conductance by muscarinic receptor stimulation was studied in the AtT-20 pituitary cell line, using the whole-cell patch-clamp technique. Only m4 mRNA was detected in these cells, thus, it is assumed that the actions of muscarinic receptor stimulation are mediated by the m4 receptor. AtT-20 cells express a slowly activating inwardly rectifying potassium conductance. Application of acetylcholine (ACh), resulted in an atropine sensitive, reversible increase in inwardly rectifying current. The ACh-induced current differed from the current recorded in control, in that it was fast activating, while the control current was slowly activating. Inclusion of GTP gamma S in the patch pipette activated an inward current with characteristics similar to the ACh-induced current, and the ACh-induced current response could be inhibited by pre-incubation with pertussis toxin (PTX). It is concluded that the m4 muscarinic receptor is coupled to an inwardly rectifying potassium conductance via a PTX sensitive G-protein.

Muscarinic receptor subtypes: modulation of ion channels.

The discovery of five muscarinic receptor subtypes by molecular genetic techniques has resulted in new approaches to understanding their function. This involves the expression of the individual genes encoding each receptor subtype in isolation, such that their effects and mechanisms of action can be studied. The coupling of the receptors with G-proteins and ion channels is the subject of this review and emphasis is placed upon the assignment of genetically defined receptor subtypes with a given physiological function. Activation of inwardly rectifying potassium conductances by m2 and m4 and inhibition by m1, as well as stimulation of calcium-dependent conductances by m1, m3 and m5 are discussed.

A large-scale, rapid public health response to rabies in an organ recipient and the previously undiagnosed organ donor.

This article describes and contrasts the public health response to two human rabies cases: one organ recipient diagnosed within days of symptom onset and the transplant donor who was diagnosed 18 months post-symptom onset. In response to an organ-transplant-related rabies case diagnosed in 2013, organ donor and recipient investigations were conducted by multiple public health agencies. Persons with potential exposure to infectious patient materials were assessed for rabies virus exposure. An exposure investigation was conducted to determine the source of the organ donor's infection. Over 100 persons from more than 20 agencies spent over 2700 h conducting contact investigations in healthcare, military and community settings. The 564 persons assessed include 417 healthcare workers [5.8% recommended for post-exposure prophylaxis (PEP)], 96 community contacts (15.6% recommended for PEP), 30 autopsy personnel (50% recommended for PEP), and 21 other persons (4.8% recommended for PEP). Donor contacts represented 188 assessed with 20.2% recommended for PEP, compared with 5.6% of 306 recipient contacts recommended for PEP. Human rabies cases result in substantial use of public health and medical resources, especially when diagnosis is delayed. Although rare, clinicians should consider rabies in cases of encephalitis of unexplained aetiology, particularly for cases that may result in organ donation.

Regulation of a family of inwardly rectifying potassium channels (Kir2) by the m1 muscarinic receptor and the small GTPase Rho.

Inwardly rectifying potassium channels Kir2.1-Kir2.3 are important regulators of membrane potential and, thus, control cellular excitability. However, little is known about the regulation of these channels. Therefore, we studied the mechanisms mediating the regulation of Kir2.1-Kir2.3 by the G-protein-coupled m1 muscarinic receptor using the whole-cell patch-clamp technique and recombinant expression in the tsA201 mammalian cell line. Stimulation of the m1 muscarinic receptor inhibited all subtypes of inward rectifier tested, Kir2.1-Kir2.3. The inhibition of each channel subtype was reversible and was attenuated by the muscarinic receptor antagonist, atropine. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) mimicked the effects of m1 receptor activation by inhibiting Kir2.1 currents. However, PMA had no effect on Kir2.2 or Kir2.3. Inclusion of 200-microM guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) in the patch pipette solution prevented the effects of m1 muscarinic receptor stimulation on all three of the channel subtypes tested, confirming the mediation of the responses by G-proteins. Cotransfection with the activated mutant of the small GTPase Rho reduced current density, while C3 exoenzyme, a selective inhibitor of Rho, attenuated the m1 muscarinic receptor-induced inhibition of Kir2.1-Kir2.3. Also, buffering the intracellular calcium concentration with a high concentration of EGTA abolished the m1 receptor-induced inhibition of Kir2.1-Kir2.3, implicating a role for calcium in these responses. These results indicate that all three of the Kir2 channels are similarly inhibited by m1 muscarinic receptor stimulation through calcium-dependent activation of the small GTPase Rho.

Effects of hyperthyroidism on delayed rectifier K+ currents in left and right murine atria.

Hyperthyroidism has been associated with atrial fibrillation (AF); however, hyperthyroidism-induced ion channel changes that may predispose to AF have not been fully elucidated. To understand the electrophysiological changes that occur in left and right atria with hyperthyroidism, the patch-clamp technique was used to compare action potential duration (APD) and whole cell currents in myocytes from left and right atria from both control and hyperthyroid mice. Additionally, RNase protection assays and immunoblotting were performed to evaluate the mRNA and protein expression levels of K(+) channel alpha-subunits in left and right atria. The results showed that 1) in control mice, the APD was shorter and the ultra-rapid delayed rectifier K(+) conductance (I(Kur)) and the sustained delayed rectifier K(+) conductance (I(ss)) were larger in the left than in the right atrium; also, mRNA and protein expression levels of Kv1.5 and Kv2.1 were higher in the left atrium; 2) in hyperthyroid mice, the APD was shortened and I(Kur) and I(ss) were increased in both left and right atrial myocytes, and the protein expression levels of Kv1.5 and Kv2.1 were increased significantly in both atria; and 3) the influence of hyperthyroidism on APD and delayed rectifier K(+) currents was more prominent in right than in left atrium, which minimized the interatrial APD difference. In conclusion, hyperthyroidism resulted in more significant APD shortening and greater delayed rectifier K(+) current increases in the right vs. the left atrium, which can contribute to the propensity for atrial arrhythmia in hyperthyroid heart.

Assisted reproduction techniques.

Many couples with infertility will require a form of assisted reproduction to achieve a pregnancy. Assisted reproduction techniques, including intrauterine insemination, gamete intrafallopian transfer and in-vitro fertilisation, are considered in this review.

Modulation of the inwardly rectifying potassium channel IRK1 by the m1 muscarinic receptor.

Modulation of the inwardly rectifying potassium channel (IRK1) by the m1 muscarinic receptor was studied with the whole-cell patch-clamp recording technique with the use of a mammalian expression system. After transfection with IRK1 and m1 muscarinic receptor genes, tsA cells expressed a cesium-sensitive inwardly rectifying potassium conductance that was reduced on application of the muscarinic receptor agonist carbachol. This reduction was reversible on washout of carbachol and could be completely inhibited by the muscarinic receptor antagonist atropine. Conversely, stimulation of the m2 muscarinic receptor, when coexpressed with IRK1, resulted in no change in IRK1 current amplitude. Phorbol-12, 13-dibutyrate, an activator of protein kinase c (PKC), mimicked the effect of m1 muscarinic receptor stimulation by inhibiting the IRK1 conductance. Preincubation with staurosporine or the specific PKC inhibitor calphostin C, before application of carbachol, fully prevented the inhibition of IRK1 by m1 muscarinic receptor stimulation. Administration of 8-bromo-cAMP, an activator of protein kinase A, and thapsigargin, a stimulator of intracellular calcium release, had no effect on IRK1, suggesting that these second messengers were not involved in the m1 muscarinic receptor-induced response. Therefore, the data indicate that the m1 muscarinic receptor inhibits IRK1, presumably via stimulation of PKC. As IRK1 is widely distributed throughout the central nervous system, it is possible that such an action on IRK1 underlies the inhibitory effects of muscarinic receptor stimulation on inwardly rectifying potassium conductances observed in the brain.

Role of the small GTPase Rho in modulation of the inwardly rectifying potassium channel Kir2.1.

The inwardly rectifying potassium channel Kir2.1 is inhibited by a variety of G-protein-coupled receptors (GPCRs). However, the mechanisms underlying the inhibition have not been fully elucidated. In this study the role of the small GTPase, Rho, in mediating this inhibition was determined. Stimulation of the m1 muscarinic receptor inhibited Kir2.1, when both receptor and channel were coexpressed in tsA201 cells. The inhibition of Kir2.1 by carbachol was reversible and atropine-sensitive. Cotransfection with a dominant-negative mutant of the small GTPase Rho abolished the inhibition of Kir2.1 with current amplitudes remaining at control levels in the presence of carbachol. Conversely, cotransfection with the constitutively activated mutant of Rho resulted in a reduction in basal Kir2.1 current amplitudes, suggesting that Rho inhibits Kir2.1. To further confirm the involvement of Rho in the signal transduction pathway, cotransfection with C3 transferase (EFC3), a selective inhibitor of Rho, abolished the reduction in Kir2.1 currents noted upon application of carbachol under control conditions. Preincubation with the phosphatidylinositol 3-kinase inhibitor wortmannin or the Rho kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2 HCl (Y-27632) had no effect on agonist-induced inhibition of Kir2.1, precluding these kinases as downstream effectors of Rho in mediation of the signal. In addition, 2'-amino-3'-methoxyflavone (PD98059), an inhibitor of mitogen-activated protein (MAP) kinase kinase (MEK), had no effect on the m1 receptor-induced inhibition of Kir2.1, suggesting that MAP kinases are not involved in the signaling pathway. In conclusion, these data indicate that the small GTPase, Rho, transduces the m1 muscarinic receptor-induced inhibition of Kir2.1 via an unidentified mechanism.

T and B cells in myelomatosis.

The majority of treated patients with myelomatosis had a significantly decreased number of T cells as measured by both lymphocyte stimulation to phytohaemagglutinin (PHA) and by their ability to form spontaneous rosettes with sheep red blood cells (SRBC). Seven untreated patients also had markedly depressed T cells. On the other hand, B-cell numbers' were significantly increased in the majority of the patients as judged by increased lymphocyte stimulation to pokeweed mitogen (PWM) and the markedly increased numbers of rosettes that were formed with antibody-coated complement activated SRBC, as well as by immunofluorescence cell markers to monospecific immunoglobulin antiserum.

Non-competitive effects of disopyramide at the neuromuscular junction: evidence for endplate ion channel block.

The effects of disopyramide were studied at the neuromuscular junction in an attempt to elucidate the mechanism of its blocking action at this site. Disopyramide 5 X 10(-5) - 10(-3) mol litre-1 produced a concentration-dependent reduction of twitch amplitude in the indirectly stimulated chick biventer cervicis preparation, but greater concentrations were required to reduce twitches elicited directly in the presence of erabutoxin-b 1 microgram ml-1. Equieffective twitch blocking doses of either disopyramide or tubocurarine greatly reduced agonist responses to acetylcholine and carbachol, but the reduction was less for magnesium-blocked twitches. Neostigmine antagonized tubocurarine-induced, but not disopyramide-induced, blockade of twitches. Concentration-response profiles to acetylcholine and carbachol were shifted to the right in a non-parallel fashion and the maximal response was depressed by disopyramide 5 X 10(-5) - 10(-4) mol litre-1. Intracellular recording studies carried out in the cut, voltage-clamped costo-cutaneous muscle of the garter snake showed that disopyramide 5 X 10(-5) - 5 X 10(-4) mol litre-1 produced a concentration- and voltage-dependent reduction of the amplitude of neurally evoked endplate-currents (EPC) and of the time constant of decay (tau) of EPC. We conclude that disopyramide possesses a non-competitive blocking action at the neuromuscular junction, which is not reversible by anticholinesterase agents. The voltage-dependent nature of the block suggests that it is mediated via blockade of the open form of the acetylcholine-activated receptor-ion channel complex.

Inhibition of the L-type calcium channel by the five muscarinic receptors (m1-m5) expressed in NIH 3T3 cells.

Modulation of L-type calcium channels by the five cloned muscarinic receptors was studied by expression of the receptors in NIH 3T3 cells. Application of acetylcholine (ACh) to cells transfected with m1-m5 resulted in a reduction in the L-type calcium current amplitude. Elevations in intracellular cAMP concentrations induced by 8-bromo-cAMP or forskolin resulted in no discernible change in the L-type calcium current. In addition, treatment with Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS), a protein kinase A (PKA) inhibitor, had no effect on the L-type currents. Conversely, application of phorbol dibutyrate, an activator of protein kinase C (PKC) or 8-bromo-cGMP, an activator of cGMP-dependent protein kinase (PKG), reduced the calcium currents. Incubation of the cells with KT5823, an inhibitor of PKG, resulted in a reduction of the response to 8-bromo-cGMP. The ACh-induced depression of L-type calcium current amplitude was sensitive to pertussis toxin (PTX) in cells transfected with the m2 or m4 receptor subtype. The m2-muscarinic-receptor-induced inhibition of the L-type calcium current was attenuated by preincubation of the cells with 8-bromo-cAMP and was unaffected by KT5823 or by calphostin C. The m1-muscarinic-receptor-induced inhibition of the L-type calcium conductance was insensitive to PTX treatment. However, the m1-induced response was blocked by preincubation of the cells with calphostin C. The present data indicate that the m2 (and possibly also the m4) muscarinic receptors inhibit the L-type calcium conductance by a reduction in cAMP concentration and that the m1 (and possibly also the m3 and m5) muscarinic receptors inhibit the L-type calcium channel via activation of PKC.