Previous studies suggest native cardiac I Kr channels are composed of alpha subunits encoded solely by the 1a transcript of the ERG1 gene. Using isoform-specific ERG1 antibodies, we have new evidence that subunits encoded by an alternate transcript, ERG1b, are also expressed in rat, canine, and human heart. The ERG1a and -1b subunits associate in vivo where they localize to the T tubules of ventricular myocytes. These data indicate native ventricular I Kr channels are heteromers containing two ␣ subunit types, ERG1a and -1b. The hERG1b-specific exon thus represents a novel target to screen for mutations causing type 2 long QT syndrome. These findings also suggest phenotypic analyses of existing type 2 long QT syndrome mutations, especially those exclusive to the hERG1a amino terminus, should be carried out in systems expressing both subunits.Long QT syndrome (LQTS) 1 is an inherited or acquired disease associated with episodic ventricular arrhythmias and sudden death. One form of inherited LQTS (LQTS-2) results from mutations in the human Ether-a-go-go-Related Gene 1 (hERG1or KCNH2) (1). hERG1 encodes a potassium channel with biophysical and pharmacological properties similar to those of cardiac I Kr , thus explaining the underlying cause of LQTS-2 as a defect in this repolarizing current (2, 3). In mammalian heart, two ERG 2 transcripts, 1a and 1b, encode proteins differing in their amino-terminal sequence (see Fig. 1A) and gating properties (4,5). Expressed in Xenopus oocytes, these subunits preferentially form heteromultimers (4). However, despite high levels of ERG1b transcript (4), first generation ERG1 antibodies against a common epitope identified only ERG1a protein in native tissue (6, 7), suggesting that ERG1b subunits do not contribute to cardiac I Kr channels. Here we provide the first direct evidence for ERG1b protein expression, localization, and co-assembly with ERG1a in cardiac ventricular myocytes. These findings indicate cardiac I Kr channels are minimally composed of ERG1a and -1b ␣ subunits. EXPERIMENTAL PROCEDURESCell Lines and Antibodies-Human embryonic kidney 293 (HEK-293) cell lines stably expressing wild-type hERG1a have been described previously (8, 9). Cell lines stably expressing hERG1a and -1b were prepared by transfection of HEK-293/hERG1a stable cells with hERG1b containing a Kozak consensus sequence (4) cloned into the BamHI/EcoRI sites of pcDNA3.1z (Invitrogen). Separate cell colonies were selected after plating cells at low density and grown in media containing 100 g/ml Zeocin, 500 g/ml neomycin for selection. All HEK-293 cells were cultured in Dulbecco's modified Eagle's medium at 37°C. The pan-ERG1 antibody, ERG1-KA, has been described previously (10). ERG1 isoform-specific antibodies were produced by Bethyl Laboratories (Montgomery, TX) in rabbits. Antisera were affinity-purified using the same peptides employed in immunization. The sequence for the ERG1b peptide is amino acids 12-25 (GALRPRAQKGRVRR), and the sequence for ERG1a is amino acids 140 -153 (SPAHDTNHRG-PPTS) (Neoclo...
Mutations in the KCNT1 (Slack, K Na 1.1) sodium-activated potassium channel produce severe epileptic encephalopathies. Expression in heterologous systems has shown that the disease-causing mutations give rise to channels that have increased current amplitude. It is not known, however, whether such gain of function occurs in human neurons, nor whether such increased K Na current is expected to suppress or increase the excitability of cortical neurons. Using genetically engineered human induced pluripotent stem cell (iPSC)-derived neurons, we have now found that sodium-dependent potassium currents are increased several-fold in neurons bearing a homozygous P924L mutation. In current-clamp recordings, the increased K Na current in neurons with the P924L mutation acts to shorten the duration of action potentials and to increase the amplitude of the afterhyperpolarization that follows each action potential. Strikingly, the number of action potentials that were evoked by depolarizing currents as well as maximal firing rates were increased in neurons expressing the mutant channel. In networks of spontaneously active neurons, the mean firing rate, the occurrence of rapid bursts of action potentials, and the intensity of firing during the burst were all increased in neurons with the P924L Slack mutation. The feasibility of an increased K Na current to increase firing rates independent of any compensatory changes was validated by numerical simulations. Our findings indicate that gain-of-function in Slack K Na channels causes hyperexcitability in both isolated neurons and in neural networks and occurs by a cell-autonomous mechanism that does not require network interactions.
Turtle auditory hair cells contain multiple isoforms of the pore‐forming α‐subunit of the large‐conductance Ca2+‐activated K+ (KCa) channel due to alternative splicing at two sites. Six splice variants were studied by expression in Xenopus oocytes. The isoforms possessed differences in apparent Ca2+ sensitivity and kinetics. The lowest Ca2+ sensitivity was observed in a novel variant resulting from a 26 amino acid deletion around one of the splice sites. Co‐expression of a bovine β‐subunit slowed the current relaxation 10‐fold compared with channels formed from α‐subunits alone but preserved the original order of kinetic differences. The β‐subunit also increased the Ca2+ sensitivity of isoforms to bring them nearer the range of sensitivity of the native KCa channels of the hair cell. With channels formed from α‐subunits or α+β‐subunits, the half‐activation voltage in a fixed Ca2+ concentration, and the time constant of the current relaxation, varied linearly with the combined size of the insertions/deletions at the splice sites. Experiments in which the β/α concentration ratio was varied indicated that the β‐subunit exerts an all‐or‐none effect on the Ca2+ sensitivity and kinetics of the channel. Co‐expression of an avian β2‐subunit had effects on kinetics and Ca2+ sensitivity of several α‐isoforms which were qualitatively similar to those produced by the bovine β‐subunit. We conclude that differential expression of alternatively spliced α‐subunit variants and a non‐uniform distribution of a β‐subunit can produce a range of KCa channel properties needed to explain the tonotopic organization of the turtle cochlea.
Alternate transcripts of the human ether-à-go-go-related gene (hERG1) encode two subunits, hERG 1a and 1b, which form potassium channels regulating cardiac repolarization, neuronal firing frequency, and neoplastic cell growth. The 1a and 1b subunits are identical except for their unique, cytoplasmic N termini, and they readily co-assemble in heterologous and native systems. We tested the hypothesis that interactions of nascent N termini promote heteromeric assembly of 1a and 1b subunits. We found that 1a and 1b N-terminal fragments bind in a direct and dose-dependent manner. hERG1 hetero-oligomerization occurs in the endoplasmic reticulum where co-expression of N-terminal fragments with hERG1 subunits disrupted oligomerization and core glycosylation. The disruption of core glycosylation, a cotranslational event, allows us to pinpoint these N-terminal interactions to the earliest steps in biogenesis. Thus, N-terminal interactions mediate hERG 1a/1b assembly, a process whose perturbation may represent a new mechanism for disease.
Defects in the trafficking of subunits encoded by the human ether-à-go-go-related gene (hERG1) can lead to catastrophic arrhythmias and sudden cardiac death due to a reduction in I Kr -mediated repolarization. Native I Kr channels are composed of two ␣ subunits, hERG 1a and 1b. In heterologous expression systems, hERG 1b subunits efficiently produce current only in heteromeric combination with hERG 1a. We used Western blot analysis and electrophysiological recordings in HEK-293 cells and Xenopus oocytes to monitor hERG 1b maturation in the secretory pathway and to determine the factors regulating surface expression of hERG 1b subunits. We found that 1b subunits expressed alone were largely retained in the endoplasmic reticulum (ER), thus accounting for the poor functional expression of homomeric 1b currents. Association with hERG 1a facilitated 1b ER export and surface expression. We show that hERG 1b subunits fail to mature because of an "RXR" ER retention signal specific to the 1b N terminus of the human sequence and not conserved in other species. Mutating the RXR facilitated maturation and functional expression of homomeric hERG 1b channels in a charge-dependent manner. Co-expression of the 1b RXR mutants with hERG 1a did not further enhance 1b maturation, suggesting that hERG 1a promotes 1b trafficking by overcoming the RXR-mediated retention. Thus, selective trafficking mechanisms regulate subunit composition of surface hERG channels.Voltage-gated potassium (K ϩ ) channels encoded by the human ether-à-go-go-related gene (hERG1 or KCNH2) mediate the repolarizing cardiac current I Kr (1, 2). Perturbation of I Kr due to mutations in the hERG1 gene or drug block of hERG 2 channels can cause sudden cardiac death associated with long QT syndrome (LQTS) (3).Native I Kr channels are composed of hERG 1a and 1b ␣ subunits encoded by alternate transcripts of the hERG1 gene (4). hERG 1a and 1b subunits have identical transmembrane and C-terminal sequences but divergent N termini (5, 6), which interact to promote heteromeric assembly early in channel biogenesis (7). When expressed heterologously, hERG 1a homomeric and 1a/1b heteromeric channels yield robust currents but with distinct properties because of the divergent N termini of the constituent subunits (5, 8, 9). However, hERG 1b homomers produce undetectable or very small currents (5). Why hERG 1b functional expression is inefficient, or how hERG 1a promotes 1b surface expression, is unknown.In this study we found that hERG 1b protein efficiently exited the ER only in the presence of hERG 1a. We tested the hypothesis that hERG 1b subunits possess ER retention/retrieval signals that are inactivated upon association with the 1a subunit, thus favoring surface expression of the heteromeric channel. Of two RXR motifs (two arginines separated by any single residue) in the 1b N terminus, surprisingly only the one motif specific to the human sequence and not conserved in other organisms subserved ER retention. Moreover, mutations within this signal promoted surface expres...
Ionotropic ␥-aminobutyric acid (GABA A and GABA C ) receptors mediate fast synaptic inhibition in the central nervous system. GABA C receptors are expressed predominantly in the retina on bipolar cell axon terminals, and are thought to mediate feedback inhibition from GABAergic amacrine cells. Utilizing the yeast two-hybrid system, we previously identified MAP1B as a binding partner of the GABA C receptor 1 subunit. Here we describe the isolation of an additional 1 interacting protein: a novel C-terminal variant of the glycine transporter GLYT-1. We show that GLYT-1 exists as four alternatively spliced mRNAs which encode proteins expressing one of two possible intracellullar N-and C-terminal domains. Variants containing the novel C terminus efficiently transport glycine when expressed in COS cells, but with unusual kinetics. We have confirmed the interaction between the novel C terminus and 1 subunit and demonstrated binding in heterologous cells. This interaction may be crucial for the integration of GABAergic and glycinergic neurotransmission in the retina.
Several mutations that cause Parkinson’s disease (PD) have been identified over the past decade. These account for 15–25% of PD cases; the rest of the cases are considered sporadic. Currently, it is accepted that PD is not a single monolithic disease but rather a constellation of diseases with some common phenotypes. While rodent models exist for some of the PD-causing mutations, research on the sporadic forms of PD is lagging due to a lack of cellular models. In our study, we differentiated PD patient-derived dopaminergic (DA) neurons from the induced pluripotent stem cells (iPSCs) of several PD-causing mutations as well as from sporadic PD patients. Strikingly, we observed a common neurophysiological phenotype: neurons derived from PD patients had a severe reduction in the rate of synaptic currents compared to those derived from healthy controls. While the relationship between mutations in genes such as the SNCA and LRRK2 and a reduction in synaptic transmission has been investigated before, here we show evidence that the pathogenesis of the synapses in neurons is a general phenotype in PD. Analysis of RNA sequencing results displayed changes in gene expression in different synaptic mechanisms as well as other affected pathways such as extracellular matrix-related pathways. Some of these dysregulated pathways are common to all PD patients (monogenic or idiopathic). Our data, therefore, show changes that are central and convergent to PD and suggest a strong involvement of the tetra-partite synapse in PD pathophysiology.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.