Activation of Wnt/ß-catenin signalling and HIF1α stabilisation alters pluripotency and differentiation/proliferation properties of human-induced pluripotent stem cells.

BACKGROUND INFORMATION: Wnt/ß-catenin signalling, in the microenvironment of pluripotent stem cells (PSCs), plays a critical role in their differentiation and proliferation. Contradictory reports on the role of Wnt/ß-catenin signalling in PSCs self-renewal and differentiation, however, render these mechanisms largely unclear. RESULTS: Wnt/ß-catenin signalling pathway in human-induced pluripotent stem cells (hiPSCs) was activated by inhibiting glycogen synthase kinase 3 (GSK3), driving the cells into a mesodermal/mesenchymal state, exhibiting proliferative, invasive and anchorage-independent growth properties, where over 70% of cell population became CD 44 (+)/CD133 (+). Wnt/ß-catenin signalling activation also altered the metabolic state of hiPSCs from aerobic glycolysis to oxidative metabolism and changed their drug and oxidative stress sensitivities. These effects of GSK3 inhibition were suppressed in HIF1α-stabilised cells. CONCLUSIONS: Persistent activation of Wnt/ß-catenin signalling endows hiPSCs with proliferative/invasive 'teratoma-like' states, shifting their metabolic dependence and allowing HIF1α-stabilisation to inhibit their proliferative/invasive properties. SIGNIFICANCE: The hiPSC potential to differentiate into 'teratoma-like' cells suggest that stem cells may exist in two states with differential metabolic and drug dependency.

Acid-Sensitive Ion Channels Are Expressed in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are potential sources for cardiac regeneration and drug development. hiPSC-CMs express all the cardiac ion channels and the unique cardiac Ca2+-signaling phenotype. In this study, we tested for expression of acid sensing ion channels (ASICs) in spontaneously beating cardiomyocytes derived from three different hiPSC lines (IMR-90, iPSC-K3, and Ukki011-A). Rapid application of solutions buffered at pH 6.7, 6.0, or 5.0 triggered rapidly activating and slowly inactivating voltage-independent inward current that reversed at voltages positive to ENa, was suppressed by 5 µM amiloride and withdrawal of [Na+]o, like neuronal ASIC currents. ASIC currents were expressed at much lower percentages and densities in undifferentiated hiPSC and in dermal fibroblasts. ASIC1 mRNA and protein were measured in first 60 days but decreased in 100 days postdifferentiation hiPSC cultures. Hyperacidification (pH 5 and 6) also triggered large Ca2+ transients in intact hiPSC-CMs that were neither ruthenium red nor amiloride-sensitive, but were absent in whole cell-clamped hiPSC-CMs. Neither ASIC1 current nor its protein was detected in rat adult cardiomyocytes, but hyperacidification did activate smaller and slowly activating currents with drug sensitivity similar to TRPV channels. Considering ASIC expression in developing but not adult myocardium, a role in heart development is likely.

CRISPR/Cas9 Gene editing of RyR2 in human stem cell-derived cardiomyocytes provides a novel approach in investigating dysfunctional Ca2+ signaling.

Type-2 ryanodine receptors (RyR2s) play a pivotal role in cardiac excitation-contraction coupling by releasing Ca2+ from sarcoplasmic reticulum (SR) via a Ca2+ -induced Ca2+ release (CICR) mechanism. Two strategies have been used to study the structure-function characteristics of RyR2 and its disease associated mutations: (1) heterologous cell expression of the recombinant mutant RyR2s, and (2) knock-in mouse models harboring RyR2 point mutations. Here, we establish an alternative approach where Ca2+ signaling aberrancy caused by the RyR2 mutation is studied in human cardiomyocytes with robust CICR mechanism. Specifically, we introduce point mutations in wild-type RYR2 of human induced pluripotent stem cells (hiPSCs) by CRISPR/Cas9 gene editing, and then differentiate them into cardiomyocytes. To verify the reliability of this approach, we introduced the same disease-associated RyR2 mutation, F2483I, which was studied by us in hiPSC-derived cardiomyocytes (hiPSC-CMs) from a patient biopsy. The gene-edited F2483I hiPSC-CMs exhibited longer and wandering Ca2+ sparks, elevated diastolic Ca2+ leaks, and smaller SR Ca2+ stores, like those of patient-derived cells. Our CRISPR/Cas9 gene editing approach validated the feasibility of creating myocytes expressing the various RyR2 mutants, making comparative mechanistic analysis and pharmacotherapeutic approaches for RyR2 pathologies possible.

Calcium signaling in human stem cell-derived cardiomyocytes: Evidence from normal subjects and CPVT afflicted patients.

Derivation of cardiomyocyte cell lines from human fibroblasts (induced pluripotent stem cells, iPSCs) has made it possible not only to investigate the electrophysiological and Ca(2+) signaling properties of these cells, but also to determine the altered electrophysiological and Ca(2+)-signaling profiles of such cells lines derived from patients expressing mutation-inducing pathologies. This approach has the potential of generating in vitro human models of cardiovascular diseases where cellular pathology can be investigated in detail and possibly specific pharmacotherapy developed. Although this approach has been applied to a number of mutations in channel proteins that cause arrhythmias, there are only few detailed reports addressing Ca(2+) signaling pathologies beyond measurements of Ca(2+) transients in intact non-voltage clamped cells. Unfortunately, full understanding of Ca(2+) signaling pathologies remains elusive, not only because of the plethora of Ca(2+) signaling proteins defects that cause arrhythmias and cardiomyopathies, but also because detailed functional properties of Ca(2+) signaling proteins are difficult to obtain. Catecholaminergic polymorphic ventricular tachycardia (CPVT1) is a malignant inherited arrhythmogenic disorder predominantly caused by mutations in the cardiac ryanodine receptor (RyR2). Thus far over 150 mutations in RyR2 have been identified that appear to cause this arrhythmia, a number of which have been expressed and studied in transgenic mice or cell-line models. The development of human iPSC-technology makes it possible to create human heart cell-lines carrying these mutations, making detailed identification of Ca(2+) signaling defects and its specific pharmacotherapy possible. In this review we shall first briefly summarize the essential characteristics of the mammalian cardiac Ca(2+) signaling, then compare them to Ca(2+) signaling phenotypes of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) and to those of rat neonatal cardiomyocytes, and categorize the possible variance in Ca(2+) signaling defects caused by different CPVT-inducing mutations as expressed in hiPSC-CMs.

Cardiac calcium signalling pathologies associated with defective calmodulin regulation of type 2 ryanodine receptor.

Cardiac ryanodine receptor (RyR2) is a homotetramer of 560 kDa polypeptides regulated by calmodulin (CaM), which decreases its open probability at diastolic and systolic Ca(2+) concentrations. Point mutations in the CaM-binding domain of RyR2 (W3587A/L3591D/F3603A, RyR2(ADA)) in mice result in severe cardiac hypertrophy, poor left ventricle contraction and death by postnatal day 16, suggesting that CaM inhibition of RyR2 is required for normal cardiac function. Here, we report on Ca(2+) signalling properties of enzymatically isolated, Fluo-4 dialysed whole cell clamped cardiac myocytes from 10-15-day-old wild-type (WT) and homozygous Ryr2(ADA/ADA) mice. Spontaneously occurring Ca(2+) spark frequency, measured at -80 mV, was 14-fold lower in mutant compared to WT myocytes. ICa, though significantly smaller in mutant myocytes, triggered Ca(2+) transients that were of comparable size to those of WT myocytes, but with slower activation and decay kinetics. Caffeine-triggered Ca(2+) transients were about three times larger in mutant myocytes, generating three- to four-fold bigger Na(+)-Ca(2+) exchanger NCX currents (INCX). Mutant myocytes often exhibited Ca(2+) transients of variable size and duration that were accompanied by similarly alternating and slowly activating INCX. The data suggest that RyR2(ADA) mutation produces significant reduction in ICa density and ICa-triggered Ca(2+) release gain, longer but infrequently occurring Ca(2+) sparks, larger sarcoplasmic reticulum Ca(2+) loads, and spontaneous Ca(2+) releases accompanied by activation of large and potentially arrhythmogenic inward INCX.

Cardiac progenitor cells engineered with Pim-1 (CPCeP) develop cardiac phenotypic electrophysiological properties as they are co-cultured with neonatal myocytes.

Stem cell transplantation has been successfully used for amelioration of cardiomyopathic injury using adult cardiac progenitor cells (CPC). Engineering of mouse CPC with the human serine/threonine kinase Pim-1 (CPCeP) enhances regeneration and cell survival in vivo, but it is unknown if such apparent lineage commitment is associated with maturation of electrophysiological properties and excitation-contraction coupling. This study aims to determine electrophysiology and Ca(2+)-handling properties of CPCeP using neonatal rat cardiomyocyte (NRCM) co-culture to promote cardiomyocyte lineage commitment. Measurements of membrane capacitance, dye transfer, expression of connexin 43 (Cx43), and transmission of ionic currents (I(Ca), I(Na)) from one cell to the next suggest that a subset of co-cultured CPCeP and NRCM becomes connected via gap junctions. Unlike NRCM, CPCeP had no significant I(Na), but expressed nifedipine-sensitive I(Ca) that could be measured more consistently with Ba(2+) as permeant ion using ramp-clamp protocols than with Ca(2+) and step-depolarization protocols. The magnitude of I(Ca) in CPCeP increased during culture (4-7 days vs. 1-3 days) and was larger in co-cultures with NRCM and with NRCM-conditioned medium, than in mono-cultured CPCeP. I(Ca) was virtually absent in CPC without engineered expression of Pim-1. Caffeine and KCl-activated Ca(2+)-transients were significantly present in co-cultured CPCeP, but smaller than in NRCM. Conversely, ATP-induced (IP(3)-mediated) Ca(2+) transients were larger in CPCeP than in NRCM. I(NCX) and I(ATP) were expressed in equivalent densities in CPCeP and NRCM. These in vitro studies suggest that CPCeP in co-culture with NRCM: a) develop I(Ca) current and Ca(2+) signaling consistent with cardiac lineage, b) form electrical connections via Cx43 gap junctions, and c) respond to paracrine signals from NRCM. These properties may be essential for durable and functional myocardial regeneration under in vivo conditions.

Hypoxic regulation of cardiac Ca2+ channel: possible role of haem oxygenase.

Acute and chronic hypoxias are common cardiac diseases that lead often to arrhythmia and impaired contractility. At the cellular level it is unclear whether the suppression of cardiac Ca(2+) channels (Ca(V)1.2) results directly from oxygen deprivation on the channel protein or is mediated by intermediary proteins affecting the channel. To address this question we measured the early effects of hypoxia (5-60 s, P(O(2)) < 5 mmHg) on Ca(2+) current (I(Ca)) and tested the involvement of protein kinase A (PKA) phosphorylation, Ca(2+)/calmodulin-mediated signalling and the haem oxygenase (HO) pathway in the hypoxic regulation of Ca(V)1.2 in rat and cat ventricular myocytes and HEK-293 cells. Hypoxic suppression of ICa) and Ca(2+) transients was significant within 5 s and intensified in the following 50 s, and was reversible. Phosphorylation by cAMP or the phosphatase inhibitor okadaic acid desensitized I(Ca) to hypoxia, while PKA inhibition by H-89 restored the sensitivity of I(Ca) to hypoxia. This phosphorylation effect was specific to Ca(2+), but not Ba(2+) or Na(+), permeating through the channel. CaMKII inhibitory peptide and Bay K8644 reversed the phosphorylation-induced desensitization to hypoxia. Mutation of CAM/CaMKII-binding motifs of the α(1c) subunit of Ca(V)1.2 fully desensitized the Ca(2+) channel to hypoxia. Rapid application of HO inhibitors (zinc protoporphyrin (ZnPP) and tin protoporphyrin (SnPP)) suppressed the channel in a manner similar to acute hypoxia such that: (1) I(Ca) and I(Ba) were suppressed within 5 s of ZnPP application; (2) PKA activation and CaMKII inhibitors desensitized I(Ca), but not I(Ba), to ZnPP; and (3) hypoxia failed to further suppress I(Ca) and I(Ba) in ZnPP-treated myocytes. We propose that the binding of HO to the CaM/CaMKII-specific motifs on Ca(2+) channel may mediate the rapid response of the channel to hypoxia.

In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells.

BACKGROUND/AIMS: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. METHODS: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. RESULTS: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca(2+) release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca(2+)-induced Ca(2+)-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. CONCLUSION: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.

c-Jun is required for TGF-ß-mediated cellular migration via nuclear Ca²âº signaling.

Tumor progression involves the acquisition of invasiveness through a basement membrane. The c-jun proto-oncogene is overexpressed in human tumors and has been identified at the leading edge of human breast tumors. TGF-ß plays a bifunctional role in tumorigenesis and cellular migration. Although c-Jun and the activator protein 1 (AP-1) complex have been implicated in human cancer, the molecular mechanisms governing cellular migration via c-Jun and the role of c-Jun in TGF-ß signaling remains poorly understood. Here, we analyze TGF-ß mediated cellular migration in mouse embryo fibroblasts using floxed c-jun transgenic mice. We compared the c-jun wild type with the c-jun knockout cells through the use of Cre recombinase. Herein, TGF-ß stimulated cellular migration and intracellular calcium release requiring endogenous c-Jun. TGF-ß mediated Ca(2+) release was independent of extracellular calcium and was suppressed by both U73122 and neomycin, pharmacological inhibitors of the breakdown of PIP(2) into IP(3). Unlike TGF-ß-mediated Ca(2+) release, which was c-Jun dependent, ATP mediated Ca(2+) release was c-Jun independent. These studies identify a novel pathway by which TGF-ß regulates cellular migration and Ca(2+) release via endogenous c-Jun.

Regulation of cardiac Ca(2+) channel by extracellular Na(+).

Hyponatremia is a predictor of poor cardiovascular outcomes during acute myocardial infarction and in the setting of preexisting heart failure [1]. There are no definitive mechanisms as to how hyponatremia suppresses cardiac function. In this report we provide evidence for direct down-regulation of Ca(2+) channel current in response to low serum Na(+). In voltage-clamped rat ventricular myocytes or HEK 293 cells expressing the L-type Ca(2+) channel, a 15mM drop in extracellular Na(+) suppressed the Ca(2+) current by ∼15%; with maximal suppression of ∼30% when Na(+) levels were reduced to 100mM or less. The suppressive effects of low Na(+) on I(Ca), in part, depended on the substituting monovalent species (Li(+), Cs(+), TEA(+)), but were independent of phosphorylation state of the channel and possible influx of Ca(2+) on Na(+)/Ca(2+) exchanger. Acidification sensitized the Ca(2+) channel current to Na(+) withdrawal. Collectively our data suggest that Na(+) and H(+) may interact with regulatory site(s) at the outer recesses of the Ca(2+) channel pore thereby directly modulating the electro-diffusion of the permeating divalents (Ca(2+), Ba(2+)).

Developmental aspects of cardiac Ca(2+) signaling: interplay between RyR- and IP(3)R-gated Ca(2+) stores.

The dominant mode of intracellular Ca(2+) release in adult mammalian heart is gated by ryanodine receptors (RyRs), but it is less clear whether inositol 1,4,5-trisphosphate (IP(3))-gated Ca(2+) release channels (IP(3)Rs), which are important during embryogenesis, play a significant role during early postnatal development. To address this question, we measured confocal two-dimensional Ca(2+) dependent fluorescence images in acutely isolated neonatal (days 1 to 2) and juvenile (days 8-10) rat cardiomyocytes, either voltage-clamped or permeabilized, where rapid exchange of solution could be used to selectively activate the two types of Ca(2+) release channel. Targeting RyRs with caffeine produced large and rapid Ca(2+) signals throughout the cells. Application of ATP and endothelin-1 to voltage-clamped, or IP(3) to permeabilized, cells produced smaller and slower Ca(2+) signals that were most prominent in subsarcolemmal regions and were suppressed by either the IP(3)R-blocker 2-aminoethoxydiphenylborate or replacement of the biologically active form of IP(3) with its L-stereoisomer. Such IP(3)R-gated Ca(2+) releases were amplified by Ca(2+)-induced Ca(2+) release (CICR) via RyRs since they were also reduced by compounds that block the RyRs (tetracaine) or deplete the Ca(2+) pools they gate (caffeine, ryanodine). Spatial analysis revealed both subsarcolemmal and perinuclear origins for the IP(3)-mediated Ca(2+) release events RyR- and IP(3)R-gated Ca(2+) signals had larger magnitudes in juvenile than in neonatal cardiomyocytes. Ca(2+) signaling was generally quite similar in atrial and ventricular cardiomyocytes but showed divergent development of IP(3)-mediated regulation in juveniles. Our data suggest that an intermediate stage of Ca(2+) signaling may be present in developing cardiomyocytes, where, in addition to RyR-gated Ca(2+) pools, IP(3)-gated Ca(2+) release is sufficiently large in magnitude and duration to trigger or contribute to activation of CICR and cardiac contraction.

L-type calcium channel as a cardiac oxygen sensor.

Acute oxygen sensing in the heart is thought to occur through redox regulation and phosphorylation of membrane channels. Here we report a novel O2-sensing mechanism involving the C-terminus of the L-type Ca2+ channel and regulated by PKA phosphorylation. In patch-clamped myocytes, oxygen deprivation decreased ICa within 40 s. The suppressive effect of anoxia was relieved by PKA-mediated phosphorylation only when Ca2+ was the charge carrier, whereas phosphorylated IBa remained sensitive to O2 withdrawal. Suppression of Ca2+ release by thapsigargin did not alter the response of ICa to anoxia, suggesting a mandatory role for Ca2+ influx and not Ca2+-induced Ca2+ release (CICR) in O2 regulation of the channel. Consistent with this idea, mutation of 80 amino acids in the Ca2+/CaM-binding domain of the recombinant alpha1C subunit that removes Ca2+ dependent inactivation (CDI) abolished O2 sensitivity of the channel. Our findings suggest that the Ca2+/CaM binding domain of the L-type Ca2+ may represent a molecular site for O2 sensing of the heart.

Local extracellular acidification caused by Ca2+-dependent exocytosis in PC12 cells.

Exocytosis of acidic synaptic vesicles may produce local extracellular acidification, but this effect has not been measured directly and its magnitude may depend on the geometry and pH-buffering capacity of both the vesicles and the extracellular space. Here we have used SNARF dye immobilized by conjugation to dextran to measure the release of protons from PC12 cells. The PC12 cells were stimulated by exposure to depolarizing K(+)-rich solution and activation was verified by fluorescence measurement of intracellular Ca(2+) and the release kinetics of GFP-labeled vesicles. Confocal imaging of the pH-dependent fluorescence from the immobile extracellular SNARF dye showed transient acidification around the cell bodies and neurites of activated PC12 cells. The local acidification was abolished when extracellular solution was devoid of Ca(2+) or strong pH-buffering was imposed with 10mM of HEPES. We conclude that the release of secretory vesicles induces local rises in proton concentrations that are co-released from synaptic vesicles with the primary neurotransmitter, and propose that the co-released protons may modulate the signaling in confined micro-domains of synapses.

Diversity of Ca2+ signaling in developing cardiac cells.

During embryonic and postnatal development, the mammalian heart undergoes rapid morphological changes with cellular differentiation that at the ultrastructural level encompasses altered expression and organization of the proteins and organelles associated with Ca(2+) signaling. Here the development and roles of the releasable Ca(2+) stores located within the sarco/endoplasmic reticulum and possibly within the nuclear envelopes are addressed. Confocal Ca(2+) imaging experiments were carried out on (i) neonatal rat cardiomyocytes, (ii) pluripotent P19 stem cells, differentiated to a cardiac phenotype by culturing with 1% dimethylsulfoxide (DMSO) in hanging droplets, and (iii) mouse embryonic cardiomyocytes isolated for short-time culture at embryonic day 9-18. The Ca(2+) release channels in neonatal and "cardiac" P19 cell were activated versus inhibited by targeting ryanodine (Ry) receptors with caffeine versus Ry and IP(3) receptors with adenosine 5'-triphosphate (ATP) or histamine versus U-73122, a phospholipase c (PLC) inhibitor. The neonatal cells displayed four recognizable phenotypes, of which two had specialized Ca(2+) stores releasable via either Ry or IP(3) receptors, and two had both types of receptors, either controlling functionally separate stores or with some degree of overlap, so that caffeine could deplete the stores releasable by ATP. The P19 cells showed variable presence of IP(3)-mediated Ca(2+) stores, and caffeine releasable stores that gained prominence in the "cardiac" phenotype, but were absent in a "neuronal" phenotype. The different roles of Ca(2+) stores were seen clearly in the mouse embryonic cells. Some cells from early stages of development (E 9-10) had Ca(2+) waves that increased in intensity during the diastolic interval and could trigger synchronous electrical excitation (via Na-Ca exchanger [NCX] and excitatory Ca(2+) and Na(+) channels). At later stages of development (E 18) we observed diastolic Ca(2+) sparks that appeared to originate from the nuclear envelope, while the Ca(2+) signals during excitation were faster and stronger in the nuclear region than in the surrounding cytoplasmic regions. However, we also found cells where the nuclear Ca(2+) signals were weaker and showed afterglow compared to the cytosolic Ca(2+) transients. We conclude that the Ca(2+) stores in cardiac cells during embryogenesis and postnatal development, that is, before the maturation of the t-tubular system and in stem cells with cardiac phenotype, show considerable diversity with respect to the pharmacology of the release channels and that regional differences in Ca(2+) signaling are observed centered in, at, and around the nucleus. We suggest that the causal relationship excitation and subcellular Ca(2+) signals in developing cardiac cells is different from that of adult cells and that the developing cardiomyocytes show a diversity that in later stages of development may be reflected in the different properties of atrial, ventricular, and pacemaker cells.

Protons enhance the gating kinetics of the alpha3/beta4 neuronal nicotinic acetylcholine receptor by increasing its apparent affinity to agonists.

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system. Although there is a vast literature on the molecular, structural and pharmacological properties of neuronal nAChR, little is known of their pH regulation. Here we report that rapid acidification (pH 6.0) enhances the current through the alpha3/beta4 recombinant nAChRs expressed stably in human embryonic kidney 293 cells and accelerates its activation kinetics without altering selectivity. Acidification also strongly accelerates the decay kinetics ("desensitization") of cytisine- and nicotine-evoked currents (pK(a) approximately 6.1), but the effect is somewhat smaller with acetylcholine and carbachol (undetermined pK(a) values), suggesting that protonation of the agonist contributes to the relaxation of the current. Transient increases of [H(+)](o) from pH 7.4 to 6.0, during the time course of decay of the current, enhances the current and accelerates its decay kinetics in a manner similar to reactivation of current by higher concentrations of agonists. We suggest that protons interact with multiple extracellular sites on alpha3/beta4 nAChRs, decreasing the effective EC(50) values of the agonist and accelerating gating kinetics, in part by promoting agonist-induced block. We speculate that corelease of protons with ACh from the secretory vesicles may induce rapid and reversible conformational changes in the slowly "desensitizing" alpha3/beta4 nAChRs, leading to accelerated signaling.