ACh Channel Research Articles

Patient-derived stem cell line UKMi005-A (hiPSC) harboring a non-synonymous heterozygous KCNJ5 gene variant

A published heterozygous gain-of-function variant in the KCNJ5 gene (p.Trp101Cys) encoding the G-protein-activated inward-rectifier potassium channel 4 subunit of the IK,ACh channel is associated with human sinus node dysfunction (SND). Differentiated hiPSC-cardiomyocytes may serve as an in-vitro model to study SND and to develop pharmacological rescue strategies. Therefore, a mutant hiPSCs line from patient-derived peripheral blood mononuclear cells (PBMCs) were reprogrammed with CytoTune-iPS 2.0 Sendai Reprogramming Kit. The hiPSC line (KCNJ5 K8) showed a regular karyotype, a typical hiPSC morphology, expressed pluripotency-associated markers in immunofluorescence stainings and RT-qPCR analysis. The ability for differentiation into all three germ layers was shown.

Effects of Ik,Ach Channel Inhibitor Tertiapin-Q on Right Atrial Preparations from Patients in Sinus Rhythm and Atrial Fibrillation

Background: The acetylcholine-activated inward rectifying current, IK,Ach, has been suggested as a novel atrial-selective putative drug target for the treatment of atrial fibrillation (AF). Here we have studied the effects of a selective IK,ACh channel inhibitor tertiapin-Q in human right atrial preparations from patients in sinus rhythm (SR) and permanent chronic atrial fibrillation (AF, >6 months).Methods: Action potentials (APs) were measured using sharp electrodes in right atrial trabeculae or patch electrodes in cardiomyocytes from patients undergoing open heart surgery (48 in SR and 34 in AF).Results and Conclusion: Tertiapin-Q (300nM) did not affect APs recorded in AF trabeculae (APD90 at 1Hz: control 205.8±9.0ms vs. drug 209.0±9.0ms, n.s.). Consistent with electrical remodelling of IK,ACh channels in AF, 1µM carbachol caused minimal effect on APD in AF trabeculae and no further effect was detected following combined exposure to carbachol and tertiapin-Q (APD90 242.0±16.9ms, 233.0±20.5ms, 234.0±19.1ms, respectively). In SR trabeculae, tertiapin-Q modestly, though not significantly, attenuated the shortening effect of carbachol on APD90: 310.0±7.7ms, 215.0±16.4ms, and 254.0±17.8ms, respectively). In the continuous presence of carbachol and tetrodotoxin (1µM), tertiapin-Q prolonged effective refractory period and APD90. In atrial cardiomyocytes from SR patients, tertiapin-Q fully reversed the carbachol-induced shortening of APD90. At a holding potential of −80mV, tertiapin-Q had no effect on the basal inward current in SR cardiomyocytes (2.0±0.5pA/pF vs. 2.5±0.7pA/pF, n=7), but significantly reduced in AF cardiomyocytes (6.8±1.1pA/pF vs. 5.9±0.8pA/pF, P<0.05, n=12), providing evidence for constitutive activity of IK,ACh in AF cardiomyocytes. From the modest effects of tertiapin-Q in multicellular preparations in comparison to cardiomyocytes, we conclude that access of the IK,ACh channel inhibitor to its target may be limited in intact superfused tissues.

Chapter Thirteen - Constitutive Activity of the Acetylcholine-Activated Potassium Current IK,ACh in Cardiomyocytes

Stimulation of the vagal nerve slows the heart rate and leads to shorter action potential duration in the atria. These effects are mainly mediated by binding of the vagal neurotransmitter acetylcholine to muscarinic type 2 receptors resulting in dissociation of Gi proteins and subsequent activation of IK,ACh-K(+) channels due to binding of Gβγ-subunits. Even though agonist-independent (constitutive) IK,ACh activity is considered negligible in the healthy heart, constitutive IK,ACh activity has been shown to contribute to remodeling processes associated with cardiac diseases such as atrial fibrillation. In this review, we summarize possible mechanisms, which may contribute to the development of constitutively active IK,ACh. For example, an increased availability of Gβγ-subunits within the IK,ACh channel complex could contribute to receptor-independent IK,ACh activation. Accordingly, reduced expression of Gα-subunits, which act as Gβγ-scavengers within the channel complex, and increased activity of nucleoside diphosphate kinases, which activate G proteins in a receptor-independent manner, are likely contributors to constitutively active IK,ACh. In addition, alterations of the IK,ACh channel composition or phosphorylation may also be involved in abnormal IK,ACh current activity. Finally, we discuss possible therapeutic applications of pharmacological IK,ACh modulators, which may represent future drug targets against cardiac diseases such as atrial fibrillation.

Riluzole blocks human muscle acetylcholine receptors

Riluzole, the only drug available against amyotrophic lateral sclerosis (ALS), has recently been shown to block muscle ACh receptors (AChRs), raising concerns about possible negative side-effects on neuromuscular transmission in treated patients. In this work we studied riluzole's impact on the function of muscle AChRs in vitro and on neuromuscular transmission in ALS patients, using electrophysiological techniques. Human recombinant AChRs composed of α(1)β(1)δ subunits plus the γ or ε subunit (γ- or ε-AChR) were expressed in HEK cells or Xenopus oocytes. In both preparations, riluzole at 0.5 μm, a clinically relevant concentration, reversibly reduced the amplitude and accelerated the decay of ACh-evoked current if applied before coapplication with ACh. The action on γ-AChRs was more potent and faster than on ε-AChRs. In HEK outside-out patches, riluzole-induced block of macroscopic ACh-evoked current gradually developed during the initial milliseconds of ACh presence. Single channel recordings in HEK cells and in human myotubes from ALS patients showed that riluzole prolongs channel closed time, but has no effect on channel conductance and open duration. Finally, compound muscle action potentials (CMAPs) evoked by nerve stimulation in ALS patients remained unaltered after a 1 week suspension of riluzole treatment. These data indicate that riluzole, while apparently safe with regard to synaptic transmission, may affect the function of AChRs expressed in denervated muscle fibres of ALS patients, with biological consequences that remain to be investigated.

K +ACh channel activation with carbachol increases atrial ANP release

Although it has been known that atrial natriuretic peptide (ANP) release is regulated through muscarinic acetylcholine receptors (mAChR), the mechanism by which this neurotransmitter regulates atrial ANP release is largely unknown. This study tested the hypothesis that K + ACh channels mediate the action of mAChR on atrial myocyte ANP release. Experiments were performed in perfused beating rabbit atria. Carbachol (CCh), an agonist of cardiac mAChR, increased atrial myocyte ANP release concomitantly with a decrease in stroke volume and intra-atrial pulse pressure in a concentration-dependent manner. Isoproterenol, a β-adrenoceptor agonist, decreased ANP release concomitantly with an increase in cAMP and mechanical dynamics. In the presence of isoproterenol, the CCh-induced increase in ANP release and decrease in cAMP efflux levels and mechanical dynamics were able to be repeated. The CCh-induced changes were blocked by selective M 2 mAChR antagonists. Tertiapin, a selective G-protein-gated K + ACh channel blocker, attenuated the CCh-induced increase in ANP release and decrease in mechanical dynamics in a concentration-dependent manner, but without a significant effect on the CCh-induced decrease in cAMP efflux levels. The CCh-induced changes in ANP release and atrial dynamics were inhibited in the atria from pertussis toxin-pretreated rabbits. These findings demonstrate that G-protein-gated K + ACh channels regulate atrial myocyte ANP release. The present study also shows that mAChR and adrenoceptors have opposing roles in the regulation of ANP release.

Changes in IK,ACh single-channel activity with atrial tachycardia remodelling in canine atrial cardiomyocytes

Although atrial tachycardia (AT) remodelling promotes agonist-independent, constitutively active, acetylcholine-regulated K+-current (I K,ACh) that increases susceptibility to atrial fibrillation (AF), the underlying changes in I K,Ach channel function are unknown. This study aimed to establish how AT remodelling affects I K,ACh single-channel function. I K,ACh single-channel activity was studied via cell-attached patch-clamp in isolated left atrial cardiomyocytes of control and AT (7 days, 400 min(-1)) dogs. Atrial tachycardia prolonged the mean duration of induced AF from 44 +/- 22 to 413 +/- 167 s, and reduced atrial effective refractory period at a 360 ms cycle length from 126 +/- 3 to 74 +/- 5 ms (n = 9/group, P < 0.001). In the absence of cholinergic stimulation, single-channel openings with typical I K,ACh conductance and rectification properties were sparse under control conditions. Atrial tachycardia induced prominent agonist-independent I K,ACh activity because of increased opening frequency (fo) and open probability (Po: approximately seven- and 10-fold, respectively, vs. control), but did not alter open time-constant, single-channel conductance, and membrane density. With maximum I K,ACh activation (10 micromol/L carbachol), channel Po was enhanced much more in control cells ( approximately 42-fold) than in AT-remodelled myocytes (approximately five-fold). The selective Kir3 current blocker tertiapin-Q (100 nmol/L) reduced fo and Po at -100 mV by 48 and 51%, respectively (P < 0.05 for each), without altering other channel properties, confirming the identity of I K,ACh. Atrial tachycardia had no significant effect on mRNA or protein expression of either of the subunits (Kir3.1, Kir3.4) underlying I K,ACh. Atrial tachycardia increases agonist-independent constitutive I K,ACh single-channel activity by enhancing spontaneous channel opening, providing a molecular basis for AT effects on macroscopic I K,ACh observed in previous studies, as well as associated refractoriness abbreviation and tertiapin-suppressible AF promotion. These results suggest an important role for constitutive I K,Ach channel opening in AT remodelling and support its interest as a potential target for AF therapy.

Tertiapin, a selective IK ACh blocker, terminates atrial fibrillation with selective atrial effective refractory period prolongation

It is well known that vagal nerve tone plays a crucial role in atrial fibrillation (AF). Acetylcholine-activated potassium current (IK ACh) mediates much of the cardiac response to vagal nerve stimulation (VNS), but the contribution of IK ACh to AF remains unknown. We investigated the role of the IK ACh channel in canine AF models using tertiapin, a selective IK ACh blocker. Tertiapin (4–41 nmol kg −1, i.v.) terminated AF in the canine VNS-induced and aconitine-induced AF models. The muscarinic M-receptor antagonist AF-DX-116 terminated AF in these models, but the adenosine receptor antagonist DPCPX had no effect. Thus it is likely that IK ACh activation via the M-receptor has a crucial role in both models. Tertiapin (12 nmol kg −1, i.v.) preferentially prolonged the atrial effective refractory period (ERP) but not the ventricular ERP under the VNS condition. This peptide (4–41 nmol kg −1, i.v.) did not affect PQ, QRS and corrected QT intervals in isoflurane-anaesthetised dogs. These results suggest that a selective IK ACh blocker is effective in canine AF models without affecting ventricular repolarisation, and might be useful for the treatment of patients with AF.

Basal activity of GIRK5 isoforms

G protein-coupled inwardly rectifying K + channels (GIRK or Kir3) form functional heterotetramers gated by Gβγ subunits. GIRK channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK5 (Kir3.5) is the oocyte homologue of the mammalian GIRK subunits that conform the K ACh channel. It has been claimed that even when the oocytes express GIRK5 proteins they do not form functional channels. However, the GIRK5 gene shows three initiation sites that suggest the existence of three isoforms. In a previous work we demonstrated the functionality of homomultimers of the shortest isoform overexpressed in the own oocytes. Remarkably, the basal GIRK5-Δ25 inward currents were not coupled to the activation of a G-protein receptor in the oocytes. These results encouraged us to study this channel in another expression system. In this work we show that Sf21 insect cells can be successfully transfected with this channel. GIRK5-Δ25 homomultimers produce time-dependent inward currents only with GTPγS in the recording pipette. Therefore, alternative modes of stimulus input to heterotrimeric G-proteins should be present in the oocytes to account for these results.

Naturally occurring mutations at the acetylcholine receptor binding site independently alter ACh binding and channel gating.

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, ɛN182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant αδ site. Studies of the analogous mutation in the δ subunit, δN187Y, disclose rate constants for ACh occupancy of the nonmutant αɛ site. The second CMS mutation, ɛD175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. ɛD175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, ɛN182Y localizes to the interface with the α subunit, and ɛD175 to the entrance of the ACh binding cavity. Both ɛN182Y and ɛD175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring ɛN182 and ɛD175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.

Nicotinic receptor fourth transmembrane domain: hydrogen bonding by conserved threonine contributes to channel gating kinetics.

The fourth transmembrane domain (M4) of the nicotinic acetylcholine receptor (AChR) contributes to the kinetics of activation, yet its close association with the lipid bilayer makes it the outermost of the transmembrane domains. To investigate mechanistic and structural contributions of M4 to AChR activation, we systematically mutated alphaT422, a conserved residue that has been labeled by hydrophobic probes, and evaluated changes in rate constants underlying ACh binding and channel gating steps. Aromatic and nonpolar mutations of alphaT422 selectively affect the channel gating step, slowing the rate of opening two- to sevenfold, and speeding the rate of closing four- to ninefold. Additionally, kinetic modeling shows a second doubly liganded open state for aromatic and nonpolar mutations. In contrast, serine and asparagine mutations of alphaT422 largely preserve the kinetics of the wild-type AChR. Thus, rapid and efficient gating of the AChR channel depends on a hydrogen bond involving the side chain at position 422 of the M4 transmembrane domain.

Isoprenaline can activate the acetylcholine-induced K+ current in canine atrial myocytes via Gs-derived betagamma subunits.

1. G protein betagamma subunits activate the acetylcholine-induced potassium current IK,ACh. There is no evidence of specificity at the level of the betagamma subunits. Therefore all G protein-coupled receptors in atrial myocytes should be able to activate IK,ACh. Paradoxically, it is often stated that isoprenaline does not activate IK,ACh. Rationales to explain this negative result include insufficient concentrations of Gs in the atrium or restricted access of Gs-derived betagamma subunits to the IK,ACh channel. We took advantage of a non-specific increase in Gs that results after infection with adenovirus. 2. Adenoviral infection unmasked a 1 microM isoprenaline-induced IK,ACh which was prevented by propranolol. Isoprenaline occasionally activated IK,ACh in uninfected and freshly dissociated atrial myocytes but the effect was larger and more consistent in infected myocytes. 3. Pertussis toxin pretreatment (100 ng ml-1 overnight) did not block the effect of isoprenaline. The effect of isoprenaline became persistent if cells were pretreated with cholera toxin (200 ng nl-1). 4. Signal transduction events distal to adenylyl cyclase were not involved in isoprenaline-induced IK,ACh. Forskolin (10 microM) did not activate IK,ACh. Inhibition of adenylyl cyclase with cytoplasmic application of 300 microM 2'-deoxyadenosine 3'-monophosphate did not prevent the activation of IK,ACh by isoprenaline. 5. Cytoplasmic application of a betagamma binding peptide derived from the C terminus of beta-adrenergic receptor kinase 1 (50 microM) prevented the effect of isoprenaline on IK,ACh. The peptide did not prevent the stimulation of the L-type calcium current by isoprenaline. 6. The results indicate that beta-adrenoceptors can activate IK,ACh in atrial myocytes through the release of betagamma subunits from Gs.

Dual effects of extracellular ATP on the muscarinic acetylcholine receptor-operated K + current in guinea-pig atrial cells

Adenosine 5′-triphosphate (ATP) is stored in sympathetic and parasympathetic nerve terminals and co-released with norepinephrine and acetylcholine during nerve stimulation. In the heart in situ parasympathetic nerve is tonically stimulated and the activated muscarinic acetylcholine-receptor-operated K + current ( I K.ACh) plays an important role in the repolarization of the atrial action potential, the sinoatrial node automaticity and the atrioventricular conduction. In the present study, effects of extracellular ATP on the I K.ACh activated by carbachol or adenosine were examined in isolated guinea-pig atrial cells by use of the patch-clamp technique. ATP (10 μM) per se produced a transient activation of I K.ACh in atrial cells held at −40 mV. When I K.ACh was preactivated by 1 μM carbachol or 10 μM adenosine, ATP (1–100 μM) produced a transient increase followed by a sustained decrease of the current. These ATP-induced biphasic changes of I K.ACh were abolished by suramin (100 μM) or reactive blue-2 (30 μM), but not by theophylline (500 μM), indicating the involvement of P 2 purinoceptors. ATP also enhanced and then partially reversed the action potential shortening induced by carbachol or adenosine in current-clamped atrial cells. Extracellular ATP did not increase but decreased the openings of the single K ACh channel that were recorded by use of a pipette solution containing 1 μM carbachol in the cell-attached mode. Thus, P 2 purinoceptor stimulation produces dual effects of ATP on the pre-activated I K.ACh and may modulate the chronotropic and inotropic responses during autonomic nerve stimulation. © 1997 Elsevier Science B.V.

Muscarinic K + Channels Are Activated by βγ Subunits and Inhibited by the GDP-Bound Form of α Subunit of Transducin

It was reported that transducin βγ subunits (T βγ) inhibited cardiac muscarinic K + (K ACh) channels activated through G K. This result has been used to support the proposal that G K activates this channel through its α subunits. Here, we have reexamined the effects of transducin subunits on K ACh channels. T βγ applied to the internal side of the inside-out patch membrane activated K ACh channels in a concentration-dependent manner (EC 50 ∼1 μM). In contrast, the GDP-bound form of transducin alpha subunits (50 nM-3 μM), but not T βγ (1 nM-1 μM), irreversibly inhibited K ACh channel openings induced either by GTP plus acetylcholine or by GTPγS (guanosine 5′-3-O-(thio)triphosphate). This inhibition was antagonized by exogenously applied T βγ (600 nM-3 μM) or brain G protein βγ subunits (10-30 nM). These data, opposite to the previous report, indicate that G K activates K ACh channels through its βγ subunits.

Actions of benzalkonium chloride as a potent depressant at the neuromuscular junction

Benzalkonium chloride (BA), a positively charged surface active agent, applied (>10 nM in the superfusing solution, produced a concentration-dependent reduction in the amplitude of both evoked endplate potentials (EPPs) and spontaneous miniature EPPs of the frog. Voltage-clamped endplate currents (EPCs) and spontaneous miniature endplate currents (MEPCs) were also diminished in amplitude, in the presence of BA (50–140 nM), whereas their time-course and reversal potential were not obviously affected. No significant change due to the addition of 50 nM BA was observed in apparent properties of the ACh channel estimated from ACh-induced current fluctuations. In the relationship between the amplitude of ACh potentials and the net charge for iontophoretic pulses at a single junction, the principal pattern of action of BA (30–60 nM) appeared to be a parallel shift to the right. At concentrations greater than about 1 μM, BA slightly increased the resting input conductance of the non-synaptic muscle fibre membrane. It was suggested that this increase was attributable to a slight increase in Cl ion conductance of the non-synaptic membrane. These results indicate that BA effectively blocked neuromuscular transmission, acting as an ACh receptor antagonist at smaller concentrations and as a more potent blocker that acts through multiple sites at greater concentrations.

Platelet-activating factor activates cardiac G K via arachidonic acid metabolites

Platelet-activating factor (PAF), added to the bathing solution, stimulated the cardiac muscarinic K + channel (K ACh) in the cell-attached patch (no agonist in the pipette). The PAF-induced K ACh channel activation was blocked by WEB2086, a PAF-receptor inhibitor, indicating that the PAF-receptor mediated the response. PAF-induced activation was prevented by nordihydroguaieretic acid, a lipoxygenase inhibitor, and AA-861, a 5-lipoxygenase inhibitor, but was not affected by indomethacin, a cyclo-oxygenase inhibitor. The PAF-induced K ACh, channel activity disappeared upon formation of inside-out patch. In this inside-out patch, intracellular GTP alone induced maximal channel reactivation, which was inhibited by GDP-βS. These results suggest that 5-lipoxygenase metabolites of PAF-released arachidonic acid cause a persistent stimulation of G K but not the K ACh channel itself, resulting in a receptor-independent activation of the K ACh channel by GTP.

Resting K conductances in pacemaker and non-pacemaker heart cells of the rabbit.

Currents through the inward rectifier K channel (iK X rec) and the ACh-operated K channel (iK X ACh) were recorded in isolated heart cells of rabbit using the patch clamp technique with electrodes having 0.5-1 micron tip inner diameter. The maximum number of overlaps of open iK X rec channels per patch was measured over 347 experiments. An average of 2.3 was found in ventricular cells and 0.03-0.06 in sinoatrial (S-A) and atrioventricular (A-V) node cells. The estimated total number of the iK X rec channels for each ventricular cell was great enough to supply the resting K conductance of the cell. The iK X ACh channel was present in S-A and A-V node cells, but was never observed in the ventricular cells. The resting conductance of the nodal cells measured with whole cell clamp recordings was about 16 times smaller than that of the ventricular cells, and was hardly decreased at all by the removal of K+ from the bath solution. Thus, the lower membrane potential of the nodal cells compared with that of the ventricular cells was attributed to the smaller K conductance of the resting membrane, which is due to the very low density of the iK X rec channel. On the other hand, the iK X ACh channel, when activated by neural regulation, may play a major role in generating the resting K conductance of the nodal cells.

A transition temperature for acetylcholine channel conductance in chick myoballs.

1. The temperature dependence of ACh channel conductance (gamma) and channel open time (tau) was determined by analysing ACh induced membrane current fluctuations in voltage clamped chick myoballs. 2. gamma decreased from 25-30 pmho at 37 degrees C to less than 5 phmo at 10 degrees C. An Arrhenius plot of gamma vs. temperature exhibited a clear break or 'transition temperature' at 20 degrees C. 3. tau increased from 2 msec at 37 degrees C to 16 msec at 10 degrees C. The Arrhenius plot of tau vs. temperature was linear. No transition temperature was detected. 4. Submicellar concentrations of the non-ionic detergent, Triton X-100 reversibly blocked ACh respnses. The effect was all-or-none at the molecular level. 5. These results are consistent with the possibility that the fluidity of membrane lipids in the ACh receptor micro-environment may influence the degree to which the channel can open.