Activation of IP3R in atrial cardiomyocytes leads to generation of cytosolic cAMP.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the inositol (1,4,5)-trisphosphate (IP3) signaling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed the fluorescence resonance energy transfer (FRET)-based cytosolic cyclic adenosine monophosphate (cAMP) sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, the addition of the α1-agonist, phenylephrine (PE, 3 µM), resulted in a FRET change of 21.20 ± 7.43%, and the addition of membrane-permeant IP3 derivative 2,3,6-tri-O-butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20 µM) resulted in a peak of 20.31 ± 6.74%. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-aminoethyl diphenylborinate (2-APB, 2.5 µM) or Xestospongin-C (0.3 µM) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3 µM) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs was not inhibited by 2-APB or Xestospongin-C. Finally, the localization of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin-C. These data support further investigation of the proarrhythmic nature and components of IP3-induced cAMP signaling to identify potential pharmacological targets.NEW & NOTEWORTHY This study shows that indirect activation of the IP3 pathway in atrial myocytes using phenylephrine and direct activation using IP3-AM leads to an increase in cAMP and is in part localized to the cell membrane. These changes can be pharmacologically inhibited using IP3R inhibitors. However, the cAMP rise in ventricular myocytes is independent of IP3R calcium release. Our data support further investigation into the proarrhythmic nature of IP3-induced cAMP signaling.

Addressing the Need for Clinical Trial End Points in Autosomal Dominant Polycystic Kidney Disease: A Report From the Polycystic Kidney Disease Outcomes Consortium (PKDOC).

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease. Expansion of multiple cysts throughout both kidneys is thought to lead to progressive loss of kidney function and kidney failure in some patients. In recent years, much has been learned about the pathophysiology of ADPKD. However, to date, only one therapy has been approved in the United States and in other regions for the treatment of ADPKD. Feasible end points and a clear regulatory pathway may stimulate further development in this area and ultimately lead to more treatments for ADPKD successfully reaching the market. In July 2016, the PKD Outcomes Consortium under the auspices of the Critical Path Institute and the PKD Foundation convened a PKD Summit to facilitate a discussion among patients, regulators, pharmaceutical sponsors, and academic clinical trialists regarding trial end points and the regulatory path to approval of new drugs for ADPKD. Following the summit, participants continued the dialogue using regularly scheduled teleconferences. This article addresses key considerations related to the design of clinical trials in ADPKD based on these discussions.

The cystic fibrosis transmembrane conductance regulator is an extracellular chloride sensor.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel that governs the quantity and composition of epithelial secretions. CFTR function is normally tightly controlled as dysregulation can lead to life-threatening diseases such as secretory diarrhoea and cystic fibrosis. CFTR activity is regulated by phosphorylation of its cytosolic regulatory (R) domain, and ATP binding and hydrolysis at two nucleotide-binding domains (NBDs). Here, we report that CFTR activity is also controlled by extracellular Cl(-) concentration ([Cl(-)]o). Patch clamp current recordings show that a rise in [Cl(-)]o stimulates CFTR channel activity, an effect conferred by a single arginine residue, R899, in extracellular loop 4 of the protein. Using NBD mutants and ATP dose response studies in WT channels, we determined that [Cl(-)]o sensing was linked to changes in ATP binding energy at NBD1, which likely impacts NBD dimer stability. Biochemical measurements showed that increasing [Cl(-)]o decreased the intrinsic ATPase activity of CFTR mainly through a reduction in maximal ATP turnover. Our studies indicate that sensing [Cl(-)]o is a novel mechanism for regulating CFTR activity and suggest that the luminal ionic environment is an important physiological arbiter of CFTR function, which has significant implications for salt and fluid homeostasis in epithelial tissues.

Interaction between 2 extracellular loops influences the activity of the cystic fibrosis transmembrane conductance regulator chloride channel.

Activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is thought to be controlled by cytoplasmic factors. However, recent evidence has shown that overall channel activity is also influenced by extracellular anions that interact directly with the extracellular loops (ECLs) of the CFTR protein. Very little is known about the structure of the ECLs or how substances interacting with these ECLs might affect CFTR function. We used patch-clamp recording to investigate the accessibility of cysteine-reactive reagents to cysteines introduced throughout ECL1 and 2 key sites in ECL4. Furthermore, interactions between ECL1 and ECL4 were investigated by the formation of disulfide crosslinks between cysteines introduced into these 2 regions. Crosslinks could be formed between R899C (in ECL4) and a number of sites in ECL1 in a manner that was dependent on channel activity, suggesting that the relative orientation of these 2 loops changes on activation. Formation of these crosslinks inhibited channel function, suggesting that relative movement of these ECLs is important to normal channel function. Implications of these findings for the effects of mutations in the ECLs that are associated with cystic fibrosis and interactions with extracellular substances that influence channel activity are discussed.

Chamber-specific contractile responses of atrial and ventricular hiPSC-cardiomyocytes to GPCR and ion channel targeting compounds: A microphysiological system for cardiac drug development.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) have found utility for conducting in vitro drug screening and disease modelling to gain crucial insights into pharmacology or disease phenotype. However, diseases such as atrial fibrillation, affecting >33 M people worldwide, demonstrate the need for cardiac subtype-specific cells. Here, we sought to investigate the base characteristics and pharmacological differences between commercially available chamber-specific atrial or ventricular hiPSC-CMs seeded onto ultra-thin, flexible PDMS membranes to simultaneously measure contractility in a 96 multi-well format. We investigated the effects of GPCR agonists (acetylcholine and carbachol), a Ca2+ channel agonist (S-Bay K8644), an HCN channel antagonist (ivabradine) and K+ channel antagonists (4-AP and vernakalant). We observed differential effects between atrial and ventricular hiPSC-CMs on contractile properties including beat rate, beat duration, contractile force and evidence of arrhythmias at a range of concentrations. As an excerpt of the compound analysis, S-Bay K8644 treatment showed an induced concentration-dependent transient increase in beat duration of atrial hiPSC-CMs, whereas ventricular cells showed a physiological increase in beat rate over time. Carbachol treatment produced marked effects on atrial cells, such as increased beat duration alongside a decrease in beat rate over time, but only minimal effects on ventricular cardiomyocytes. In the context of this chamber-specific pharmacology, we not only add to contractile characterization of hiPSC-CMs but propose a multi-well platform for medium-throughput early compound screening. Overall, these insights illustrate the key pharmacological differences between chamber-specific cardiomyocytes and their application on a multi-well contractility platform to gain insights for in vitro cardiac liability studies and disease modelling.

Incomplete incorporation of tandem subunits in recombinant neuronal nicotinic receptors.

Tandem constructs are increasingly being used to restrict the composition of recombinant multimeric channels. It is therefore important to assess not only whether such approaches give functional channels, but also whether such channels completely incorporate the subunit tandems. We have addressed this question for neuronal nicotinic acetylcholine receptors, using a channel mutation as a reporter for subunit incorporation. We prepared tandem constructs of nicotinic receptors by linking alpha (alpha2-alpha4, alpha6) and beta (beta2, beta4) subunits by a short linker of eight glutamine residues. Robust functional expression in oocytes was observed for several tandems (beta4_alpha2, beta4_alpha3, beta4_alpha4, and beta2_alpha4) when coexpressed with the corresponding beta monomer subunit. All tandems expressed when injected alone, except for beta4_alpha3, which produced functional channels only together with beta4 monomer and was chosen for further characterization. These channels produced from beta4_alpha3 tandem constructs plus beta4 monomer were identical with receptors expressed from monomer alpha3 and beta4 constructs in acetylcholine sensitivity and in the number of alpha and beta subunits incorporated in the channel gate. However, separately mutating the beta subunit in either the monomer or the tandem revealed that tandem-expressed channels are heterogeneous. Only a proportion of these channels contained as expected two copies of beta subunits from the tandem and one from the beta monomer construct, whereas the rest incorporated two or three beta monomers. Such inaccuracies in concatameric receptor assembly would not have been apparent with a standard functional characterization of the receptor. Extensive validation is needed for tandem-expressed receptors in the nicotinic superfamily.

Prevention and partial reversal of diabetes-induced changes in enteric nerves of the rat ileum by combined treatment with alpha-lipoic acid and evening primrose oil.

Treatment with alpha-lipoic acid (LA) or evening primrose oil (EPO), individually, fails to prevent diabetes-induced changes in enteric nerves. Since synergy between these treatments has been reported, the aim was to investigate the effectiveness of combined LA/EPO treatment. LA and EPO were administered in the diet (approximately 80 and 200 mg/kg/day, respectively) to control and diabetic (induced by streptozotocin, 65 mg/kg, i.p.) rats. For prevention, treatment started after 1 week and lasted 7 weeks. For reversal, treatment lasted 4 weeks and was initiated after 8 weeks. Nerves supplying the ileum containing vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and noradrenaline (NA) were examined immunohistochemically or biochemically. Diabetes caused a significant increase in VIP-containing cell bodies (p<0.001), decrease in NA content (p<0.01) and loss of CGRP-immunoreactivity. LA/EPO treatment totally prevented diabetes-induced changes in VIP (p<0.001) and CGRP and partially reversed (p<0.05) these changes once they had been allowed to develop. In contrast, treatment had no effect on diabetes-induced changes in NA-containing nerves. Therefore, LA and EPO are only effective at treating diabetes-induced changes in some enteric nerves when administered in combination. However, diabetes-induced changes in NA-containing nerves are resistant to treatment.

Human α3ß4 neuronal nicotinic receptors show different stoichiometry if they are expressed in Xenopus oocytes or mammalian HEK293 cells.

BACKGROUND: The neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the α3 and ß4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the α5 and/or ß2 subunits. We compared the properties of human α3ß4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and α:ß subunit ratio. METHODOLOGY/PRINCIPAL FINDINGS: Two distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the α subunit, as reported for α4ß2 channels. This interpretation is supported by the pattern of change in acetylcholine (ACh) sensitivity observed when a hydrophilic Leu to Thr mutation was inserted in position 9' of the second transmembrane domain, as the effect of mutating the more abundant subunit is greater. Unlike α4ß2 channels, for α3ß4 receptors the putative two-α form is the predominant one in oocytes (at 1:1 α:ß cRNA ratio). This two-α form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-α form is the predominant one in HEK cells transfected with a 1:1 α:ß DNA ratio or in oocytes at 9:1 α:ß RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-α form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-α form gave rise to short bursts (14 ms) of high conductance (39 pS). CONCLUSIONS/SIGNIFICANCE: Like other neuronal nicotinic receptors, the α3ß4 receptor can exist in two different stoichiometries, depending on whether it is expressed in oocytes or in mammalian cell lines and on the ratio of subunits transfected.

Constraining the expression of nicotinic acetylcholine receptors by using pentameric constructs.

Much of our understanding of ligand-gated ion channels comes from heterologous expression studies. However, this technique cannot produce receptors with a predetermined subunit composition for channels formed by several different subunits and cannot insert a single mutation copy if the subunit of interest is present in several copies in the channel. Here, we describe a novel approach that overcomes these problems by expressing pentameric constructs, in which the code of the five subunits is linked (i.e., beta4_beta4_alpha3_beta4_alpha3). This is the first time that a concatemer of the complete pentameric receptor has been expressed for channels in the cysteine-loop superfamily. The presence of the linker did not change the agonist or antagonist sensitivity of alpha3beta4 nicotinic receptors. We show evidence that the expressed receptors were made up of alpha3 and beta4 subunits in one pentameric fusion protein as designed in the construct. This approach can be applied to any nicotinic superfamily receptor to produce channels with a defined subunit arrangement and to introduce specific mutations at any desired location of the pentameric fusion protein.

Incorporation of the beta3 subunit has a dominant-negative effect on the function of recombinant central-type neuronal nicotinic receptors.

The beta3 neuronal nicotinic subunit is localized in dopaminergic areas of the central nervous system, in which many other neuronal nicotinic subunits are expressed. So far, beta3 has only been shown to form functional receptors when expressed together with the alpha3 and beta4 subunits. We have systematically tested in Xenopus laevis oocytes the effects of coexpressing human beta3 with every pairwise functional combination of neuronal nicotinic subunits likely to be relevant to the central nervous system. Expression of alpha7 homomers or alpha/beta pairs (alpha2, alpha3, alpha4, or alpha6 together with beta2 or beta4) produced robust nicotinic currents for all combinations, save alpha6beta2 and alpha6beta4. Coexpression of wild-type beta3 led to a nearly complete loss of function (measured as maximum current response to acetylcholine) for alpha7 and for all functional alpha/beta pairs except for alpha3beta4. This effect was also seen in hippocampal neurons in culture, which lost their robust alpha7-like responses when transfected with beta3. The level of surface expression of nicotinic binding sites (alpha3beta4, alpha4beta2, and alpha7) in tsA201 cells was only marginally affected by beta3 expression. Furthermore, the dominant-negative effect of beta3 was abolished by a valine-serine mutation in the 9' position of the second transmembrane domain of beta3, a mutation believed to facilitate channel gating. Our results show that incorporation of beta3 into neuronal nicotinic receptors other than alpha3beta4 has a powerful dominant-negative effect, probably due to impairment in gating. This raises the possibility of a novel regulatory role for the beta3 subunit on neuronal nicotinic signaling in the central nervous system.

The effects of beta3 subunit incorporation on the pharmacology and single channel properties of oocyte-expressed human alpha3beta4 neuronal nicotinic receptors.

We compared the main properties of human recombinant alpha3beta4beta3 neuronal nicotinic receptors with those of alpha3beta4 receptors, expressed in Xenopus oocytes. beta3 incorporation decreased the channel mean open time (from 5.61 to 1.14 ms, after approximate correction for missed gaps) and burst length. There was also an increase in single channel slope conductance from 28.8 picosiemens (alpha3beta4) to 46.7 picosiemens (alpha3beta4beta3; in low divalent external solution). On the other hand, the calcium permeability (determined by a reversal potential method in chloride-depleted oocytes) and the pharmacological properties of beta3-containing receptors differed little from those of alpha3beta4. The main pharmacological difference in alpha3beta4beta3 "triplet" receptors was a 3-fold decrease in the potency of lobeline relative to acetylcholine. Nevertheless, there was no change in the rank order of potency for agonists (epibatidine >> lobeline > cytisine, 1,1-dimethyl-4-phenylpiperazinium iodide, nicotine > acetylcholine > carbachol for both receptors; measured at low agonist concentrations). Sensitivity to the competitive antagonists trimetaphan (0.2-1 microM) and dihydro-beta-erythroidine (30 microM) was similar for the two combinations, with a Schild KB for trimetaphan of 76 and 66 nM on alpha3beta4 and alpha3beta4beta3, respectively. The change in single channel conductance confirms that beta3 replaces a beta4 subunit in the pentamer. The absence of pronounced differences in the pharmacological profile of the triplet receptor argues against a role for the beta3 subunit in the formation of agonist binding sites, whereas the changes in channel kinetics suggest an important effect on receptor gating. The shortening of the burst length of beta3-containing receptors implies that any synaptic currents mediated by such channels would have faster decay kinetics.