The ERG1A K+ channel modulates the protein degradation that contributes to skeletal muscle atrophy by increasing intracellular calcium concentration ([Ca2+]i) and enhancing calpain activity, but the mechanism by which the channel regulates the [Ca2+]i is not known. Here, we have investigated the effect of human ERG1A (HERG) on [Ca2+]i in C2C12 myotubes, using Fura-2 calcium assays, immunoblot, RT-qPCR, and electrophysiology. The data show that the rise in [Ca2+]i induced by KCl-stimulated depolarization is of greater amplitude in C2C12 myotubes over-expressing HERG relative to controls, but this difference does not result from an increase in L-type channel (Cav1.1) Ca2+ influx because there is no statistical difference in the nifedipine-sensitive response upon depolarization between the expression groups. Indeed, HERG overexpression in C2C12 myotubes has no effect on the amplitude of L-type channel current nor does it affect the mRNA levels nor protein abundance of the Cav1.1 channel. This finding suggests that HERG modulates excitation coupled calcium entry (ECCE). Indeed, the HERG-enhanced increase in [Ca2+]i induced by depolarization is blocked by 2-aminoethoxydiphenyl borate, an inhibitor of ECCE. Further, HERG also modulates the activity of ryanodine receptors (RYR1, a component of ECCE) as well as store operated calcium entry (SOCE). Therefore, we investigated the effect of HERG on calsequestrin1, a calcium buffering/binding protein known to modulate RYR1 and SOCE activities. Indeed, we find that calsequestrin1 mRNA levels are decreased 0.83-fold (p < 0.05) and the total protein abundance is lowered 77% (p < 0.05) in myotubes over-expressing HERG relative to controls. In conclusion, the data show that ERG1A overexpression modulates [Ca2+]i in skeletal muscle cells by lowering the abundance of the calcium buffering/binding protein calsequestrin1 which interacts with RyR1 and SOCE pathways. Indeed, we report that overexpression of HERG in myotubes increases [Ca2+]i by modulation of RyR1 as well as ECCE and SOCE activities. It is likely that HERG enhancement of RyR1 activity, through decreased Casq1 abundance, is increasing [Ca2+]i. This study provides a potential mechanism to explain how upregulation of ERG1A contributes to increased [Ca2+]i and, thus, atrophy in skeletal muscle.

The ER Ca2+ channel ryanodine receptor 2 (RyR2) is required for maintenance of insulin content and glucose-stimulated insulin secretion, in part, via regulation of the protein IRBIT in the insulinoma cell line INS-1. Here, we examined store-operated and depolarization-dependent Ca2+entry using INS-1 cells in which either RyR2 or IRBIT were deleted. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but was unchanged in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and stimulated (500 μM carbachol) phospholipase C (PLC) activity was also reduced specifically in RyR2KO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but was potentiated by an EPAC-selective cAMP analog in all three cell lines. Cellular PIP2 levels were increased and cortical f-actin levels were reduced in RyR2KO cells compared to controls. Whole-cell Cav channel current density was increased in RyR2KO cells compared to controls, and barium current was reduced by acute activation of the lipid phosphatase pseudojanin preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates β-cell electrical activity by controlling Cav current density and SK channel activation.

The role of ER Ca 2+ release via ryanodine receptors (RyR) in pancreatic β-cell function is not well defined. Deletion of RyR2 from the rat insulinoma INS-1 (RyR2 KO ) enhanced IP 3 receptor activity stimulated by 7.5 mM glucose, coincident with reduced levels of the protein I P 3 R eceptor B inding protein released with I nositol 1,4,5 T risphosphate (IRBIT). Insulin content, basal (2.5 mM glucose) and 7.5 mM glucose-stimulated insulin secretion were reduced in RyR2 KO and IRBIT KO cells compared to controls. INS2 mRNA levels were reduced in both RyR2 KO and IRBIT KO cells, but INS1 mRNA levels were specifically decreased in RyR2 KO cells. Nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in RyR2 KO and IRBIT KO cells. DNA methylation of the INS1 and INS2 gene promotor regions was very low, and not different among RyR2 KO , IRBIT KO , and controls, but exon 2 of the INS1 and INS2 genes was more extensively methylated in RyR2 KO and IRBIT KO cells. Exploratory proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. These results suggest that RyR2 regulates IRBIT levels and activity in INS-1 cells, and together maintain insulin content and secretion, and regulate the proteome, perhaps via DNA methylation.

BACKGROUND:The potassium channel encoded by the ether-a-gogo-related gene 1A (erg1a) has been detected in the atrophying skeletal muscle of mice experiencing either muscle disuse or cancer cachexia and further evidenced to contribute to muscle deterioration by enhancing ubiquitin proteolysis; however, to our knowledge, ERG1A has not been reported in human skeletal muscle.METHODS AND RESULTS:Here, using immunohistochemistry, we detect ERG1A immunofluorescence in human Rectus abdominis skeletal muscle sarcolemma. Further, using single point brightness data, we report the detection of ERG1A immunofluorescence at low levels in the Rectus abdominis muscle sarcolemma of young adult humans and show that it trends toward greater levels (10.6%) in healthy aged adults. Interestingly, we detect ERG1A immunofluorescence at a statistically greater level (53.6%; p < 0.05) in the skeletal muscle of older cancer patients than in age-matched healthy adults. Importantly, using immunoblot, we reveal that lower mass ERG1A protein is 61.5% (p < 0.05) more abundant in the skeletal muscle of cachectic older adults than in healthy age-matched controls. Additionally, we report that the ERG1A protein is detected in a cultured human rhabdomyosarcoma line that may be a good in vitro model for the study of ERG1A in muscle.CONCLUSIONS:The data demonstrate that ERG1A is detected more abundantly in the atrophied skeletal muscle of cancer patients, suggesting it may be related to muscle loss in humans as it has been shown to be in mice experiencing muscle atrophy as a result of malignant tumors.

The ERG1A potassium channel alternative splice variant is detected at low abundance in normal skeletal muscle; however, it is up-regulated in atrophying skeletal muscle, where it has been shown to modulate both intracellular calcium levels and ubiquitin proteasome proteolysis (UPP). The pathways by which this modulation occurs are not known. Therefore, we transduced C2C12 myotubes with either an adenovirus encoding HERG or an appropriate control virus (n=6). At 48 hours after viral treatments, we extracted total RNA from these cells and reverse transcribed them into cDNA, selecting for coding sequences (i.e., mRNA) by using poly(T) oligomers. The cDNA libraries were sequenced on Illumina's NovaSeq platform and sequence quality was assessed using FastQC (v 0.11.7) for all samples. Quality trimming was performed with the FASTX-Toolkit (v 0.0.14) to remove bases with a Phred33 score of less than 30. The resulting reads of at least 50 bases were mapped against the reference genome using STAR. The mapping results and the annotation file for the reference genome were used as input for HTSeq7 (v 0.7.0) to obtain read counts. Counts from all replicates were merged together to produce a read count matrix for all samples and this count matrix was used for downstream differential gene expression analysis (DGEA). DGEA between treatment and control was carried out using ‘R’ (v 3.5.1). The data demonstrate that HERG expression does produce numerous changes in the gene expression profile of C2C12 myotubes. Indeed, we find that HERG potentially modulates expression of numerous genes (see Table) connected with skeletal muscle atrophy, specifically ubiquitin proteasome proteolysis and with the cytokine interferon, which has been connected with muscle atrophy. The data suggest that HERG does play a role in modulation of protein degradation in skeletal muscle.

IntroductionA heteromultimer of the ERG1a/1b potassium channel is known to contribute to repolarization of the cardiac action potential. A homomultimer of the ERG1a subunit has been detected in the atrophying skeletal muscle of mice experiencing muscle disuse and cancer cachexia and has been shown to contribute to muscle atrophy by enhancing ubiquitin proteolysis; however, to our knowledge, ERG1 has not been reported in human skeletal muscle.Methods and ResultsHere, using immunohistochemistry we detect ERG1 immunofluorescence at low levels in Rectus abdominis muscle of young adult humans and show that it trends toward greater levels (10.6%) in the same muscle of healthy aged adults. Further, we detect ERG1 immunofluorescence at a statistically greater level (53.6%; p<0.05) in the Rectus abdominis muscle of older people having cancer cachexia than in age‐matched adults. Additionally, we observe ERG1 immunofluorescence in skeletal muscle sarcolemma and detect that its fluorescent pattern is consistent with I‐band localization.DiscussionThe data suggest that ERG1 may be related to muscle loss in humans and may be located in t‐tubules where it could influence calcium handling.Support or Funding InformationThis work was supported in part by Southern Illinois University School of Medicine (Research Seed Grant to ALP). The work was also supported in part by the Italian Society for Cancer Research (AIRC grant number 17388 to M Sandri) and by Austrian national co‐financing of the Austrian Federal Ministry of Science and Research; Ludwig Boltzmann Society (Vienna, Austria) to H Kern. PK would like to acknowledge National Science Foundation (CHE 0748676), NIH (GM 106364) for partial financial support of this research. The authors wish to thank Dr. Don Caspary who graciously provided FBN rat muscles. Human Pectoralis minor muscle samples were provided by the SIU School of Medicine Tissue Bank, a Simmons Cancer Institute funded program. Other investigators may have received samples from these same tissue specimens. UC thanks the IRCCS Fondazione Ospedale San Camillo, Venezia, Italy and the A&C M‐C Foundation for Translational Research for scientific support.

Background: The ERG1a potassium channel has been detected in the atrophying skeletal muscle of mice experiencing either muscle disuse or cancer cachexia and further evidenced to contribute to muscle deterioration by enhancing ubiquitin proteolysis; however, to our knowledge, ERG1 has not been reported in human skeletal muscle. Methods and Results: Here, using immunohistochemistry, we detect ERG1 immunofluorescence in human Rectus abdominis skeletal muscle sarcolemma. Further, using single point brightness data, we report detection of ERG1 immunofluorescence at low levels in the Rectus abdominis muscle sarcolemma of young adult humans and show that it trends toward greater levels (10.6%) in healthy aged adults. Interestingly, we detect ERG1 immunofluorescence at a statistically greater level (53.6%; p<0.05) in the skeletal muscle of older people having cancer cachexia than in age-matched adults. Importantly, using immunoblot, we reveal that ERG1 protein is 38% (p<0.09) more abundant in the skeletal muscle of cachectic older adults than in healthy age-matched controls. Additionally, we report that the ERG1 fluorescent pattern is consistent with I-band localization. Conclusions: The data suggest that ERG1 may be related to muscle loss in humans and is located in t-tubules where it could influence calcium handling.

Background Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological therapy for atrophy; therefore, exploration of the mechanisms contributing to atrophy is essential because it will eventually lead to discovery of an effective therapeutic target. The ether - a - go - go related gene ( ERG1A ) K + channel has been shown to contribute to atrophy by upregulating ubiquitin proteasome proteolysis in cachectic and unweighted mice and has also been implicated in calcium modulation in cancer cells. Methods We transduced C 2 C 12 myotubes with either a human ERG1A encoded adenovirus or an appropriate control virus. We used fura-2 calcium indicator to measure intracellular calcium concentration and Calpain-Glo assay kits (ProMega) to measure calpain activity. Quantitative PCR was used to monitor gene expression and immunoblot evaluated protein abundances in cell lysates. Data were analyzed using either a Student’s t test or two-way ANOVAs and SAS software as indicated. Results Expression of human ERG1A in C 2 C 12 myotubes increased basal intracellular calcium concentration 51.7% ( p < 0.0001; n = 177). Further, it increased the combined activity of the calcium-activated cysteine proteases, calpain 1 and 2, by 31.9% ( p < 0.08; n = 24); these are known to contribute to degradation of myofilaments. The increased calcium levels are likely a contributor to the increased calpain activity; however, the change in calpain activity may also be attributable to increased calpain protein abundance and/or a decrease in levels of the native calpain inhibitor, calpastatin. To explore the enhanced calpain activity further, we evaluated expression of calpain and calpastatin genes and observed no significant differences. There was no change in calpain 1 protein abundance; however, calpain 2 protein abundance decreased 40.7% ( p < 0.05; n = 6). These changes do not contribute to an increase in calpain activity; however, we detected a 31.7% decrease ( p < 0.05; n = 6) in calpastatin which could contribute to enhanced calpain activity. Conclusions Human ERG1A expression increases both intracellular calcium concentration and combined calpain 1 and 2 activity. The increased calpain activity is likely a result of the increased calcium levels and decreased calpastatin abundance.

In skeletal muscle increased intracellular calcium concentration ([Ca2+]i; uM range) is necessary for excitation‐contraction coupling; however, smaller increases in [Ca2+]i (nM range) can modulate other physiological processes in non‐contracting muscle. Indeed, fluctuations in localized calcium concentration can serve as a second messenger. In contrast, inappropriate changes in [Ca2+]i can have detrimental effects on muscle tissue and are associated with numerous skeletal muscle pathologies, for example muscular dystrophy, amyotrophic lateral sclerosis, malignant hyperthermia, cancer cachexia, and atrophy. Therefore, [Ca2+]i must be tightly regulated in terms of time, space and amplitude for cellular processes to occur in a properly coordinated fashion. In earlier reports, we showed that the ERG1a K+ channel is upregulated in atrophying skeletal muscle and contributes to increased ubiquitin proteasome proteolysis. Here, using the ratiometric Ca2+ indicator fura‐2AM, we show that ERG1a expression in C2C12 myotubes produces an increase in basal [Ca2+]i as well as a transient increase in [Ca2+]i as a consequence of depolarization. We explored this transient increase in [Ca2+]i using pharmacological agents. The data demonstrate that the ERG1a‐induced increase is not sensitive to the L‐type calcium channel blocker nifedipine, suggesting that it does not result from modulation of Cav1.1 channels. To further support this data, immunoblots reveal that there is no change in Cav1.1 channel abundance. However, the data do demonstrate that the increase in [Ca2+]i is sensitive to the SERCA blocking agent thapsigargin, suggesting that the source of the calcium is the sarcoplasmic reticulum stores. Additionally, the data reveal that ERG1a expression also increases basal calpain activity. In summary, to date the data show that ERG1a increases [Ca2+]i levels and suggest that this increase could occur, at least in part, as a result of release of calcium from sarcoplasmic reticulum stores. Additionally, the data reveal that ERG1a expression also increases calpain activity, suggesting that the increase in intracellular calcium results in increased calpain activity and possibly contributes to the increased proteolysis that occurs in atrophic skeletal muscle.Support or Funding InformationU.S. Department of Defense, PRMRP Grant# PR170326This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pancreatic β-cells express multiple phosphodiesterase (PDE) subtypes, but the specific roles for each in β-cell function, particularly in humans, is not clear. We evaluated the cellular role of PDE1, PDE3, and PDE4 activity in the rat insulinoma cell line INS-1 and in primary human β-cells using subtype-selective PDE inhibitors. Using a genetically encoded, FRET-based cAMP sensor, we found that the PDE1 inhibitor 8MM-IBMX, elevated cAMP levels in the absence of glucose to a greater extent than either the PDE3 inhibitor cilostamide or the PDE4 inhibitor rolipram. In 18 mM glucose, PDE1 inhibition elevated cAMP levels to a greater extent than PDE3 inhibition in INS-1 cells, while PDE4 inhibition was without effect. Inhibition of PDE1 or PDE4, but not PDE3, potentiated glucose-stimulated insulin secretion in INS-1 cells. PDE1 inhibition, but not PDE3 or PDE4 inhibition, reduced palmitate-induced caspase-3/7 activation, and enhanced CREB phosphorylation in INS-1 cells. In human β-cells, only PDE3 or PDE4 inhibition increased cAMP levels in 1.7 mM glucose, but PDE1, PDE3, or PDE4 inhibition potentiated cAMP levels in 16.7 mM glucose. Inhibition of PDE1 or PDE4 increased cAMP levels to a greater extent in 16.7 mM glucose than in 1.7 mM glucose in human β-cells. In contrast, elevation of cAMP levels by PDE3 inhibition was not different at these glucose concentrations. PDE1 inhibition also potentiated insulin secretion from human islets, suggesting that the role of PDE1 may be conserved between INS-1 cells and human pancreatic β-cells. Our results suggest that inhibition of PDE1 may be a useful strategy to potentiate glucose-stimulated insulin secretion, and to protect β-cells from the toxic effects of excess fatty acids.

β-Hydroxy difluoromethyl ketones represent the newest class of agonists of the GABA-B receptor, and they are structurally distinct from all other known agonists at this receptor because they do not display the carboxylic acid or amino group of γ-aminobutyric acid (GABA). In this report, the design, synthesis, and biological evaluation of additional analogues of β-hydroxy difluoromethyl ketones characterized the critical nature of the substituted aromatic group on the lead compound. The importance of these new data is interpreted by docking studies using the X-ray structure of the GABA-B receptor. Moreover, we also report that the synthesis and biological evaluation of β-amino difluoromethyl ketones provided the most potent compound across these two series.

Nifedipine and FPL 64176 (FPL), which block and potentiate L-type voltage-gated Ca2+ channels, respectively, modulate Ca(v)1.2 more potently than Ca(v)1.3. To identify potential strategies for developing subtype-selective inhibitors, we investigated the role of divergent amino acid residues in transmembrane domains IIIS5 and the extracellular IIIS5-3P loop region in modulation of these channels by nifedipine and FPL. Insertion of the extracellular IIIS5-3P loop from Ca(v)1.2 into Ca(v)1.3 (Ca(v)1.3+) reduced the IC50 of nifedipine from 289 to 101 nM, and substitution of S1100 with an A residue, as in Ca(v)1.2, accounted for this difference. Substituting M1030 in IIIS5 to V in Ca(v)1.3+ (Ca(v)1.3+V) further reduced the IC50 of nifedipine to 42 nM. FPL increased current amplitude with an EC50 of 854 nM in Ca(v)1.3, 103 nM in Ca(v)1.2, and 99 nM in Ca(v)1.3+V. In contrast to nifedipine block, substitution of M1030 to V in Ca(v)1.3 had no effect on potency of FPL potentiation of current amplitude, but slowed deactivation in the presence and absence of 10 mu M FPL. FPL had no effect on deactivation of Ca(v)1.3/dihydropyridine-insensitive (DHPi), a channel with very low sensitivity to nifedipine block (IC50 similar to 93 mu M), but did shift the voltage-dependence of activation by similar to -10 mV. We conclude that the MN variation in IIIS5 and the S/A variation in the IIIS5-3P loop of Ca(v)1.2 and Ca(v)1.3 largely determine the difference in nifedipine potency between these two channels, but the difference in FPL potency is determined by divergent amino acids in the IIIS5-3P loop.

Nifedipine and FPL 64176 (FPL), which block and potentiate L-type voltage-gated Ca2+ channels, respectively, modulate Cav1.2 more potently than Cav1.3. To identify potential strategies for developing subtype-selective inhibitors, we investigated the role of divergent amino acid residues in transmembrane domains IIIS5 and the extracellular IIIS5-3P loop region in modulation of these channels by nifedipine and FPL. Insertion of the extracellular IIIS5-3P loop from Cav1.2 into Cav1.3 (Cav1.3+) reduced the IC50 of nifedipine from 289 to 101 nM, and substitution of S1100 with an A residue, as in Cav1.2, accounted for this difference. Substituting M1030 in IIIS5 to V in Cav1.3+ (Cav1.3+V) further reduced the IC50 of nifedipine to 42 nM. FPL increased current amplitude with an EC50 of 854 nM in Cav1.3, 103 nM in Cav1.2, and 99 nM in Cav1.3+V. In contrast to nifedipine block, substitution of M1030 to V in Cav1.3 had no effect on potency of FPL potentiation of current amplitude, but slowed deactivation in the presence and absence of 10 μM FPL. FPL had no effect on deactivation of Cav1.3/dihydropyridine-insensitive (DHPi), a channel with very low sensitivity to nifedipine block (IC50 ∼93 μM), but did shift the voltage-dependence of activation by ∼−10 mV. We conclude that the M/V variation in IIIS5 and the S/A variation in the IIIS5-3P loop of Cav1.2 and Cav1.3 largely determine the difference in nifedipine potency between these two channels, but the difference in FPL potency is determined by divergent amino acids in the IIIS5-3P loop.

beta-Hydroxy difluoromethyl ketones represent the newest class of agonists of the GABA-B receptor, and they are structurally distinct from all other known agonists at this receptor because they do not display the carboxylic acid or amino group of gamma-aminobutyric acid (GABA). In this report, the design, synthesis, and biological evaluation of additional analogues of beta-hydroxy difluoromethyl ketones characterized the critical nature of the substituted aromatic group on the lead compound. The importance of these new data is interpreted by docking studies using the X-ray structure of the GABA-B receptor. Moreover, we also report that the synthesis and biological evaluation of beta-amino difluoromethyl ketones provided the most potent compound across these two series. (C) 2018 Elsevier Ltd. All rights reserved.

Skeletal muscle atrophy is the loss of muscle size and strength caused by an imbalance in protein degradation and protein synthesis. It occurs with normal aging, neural and skeletal muscle injuries, and with diseases such as diabetes, cancer cachexia, AIDS, Muscular Dystrophy, etc. If unabated, skeletal muscle atrophy can be very debilitating and even cause death. Three proteolytic systems are known to be upregulated in atrophic muscle: 1) ubiquitin proteasome proteolysis (UPP); 2) calcium activated calpains; and 3) lysosomal cathepsins. HERG1 is a K + ion channel shown to be up‐regulated in atrophying skeletal muscle. We have shown that it transiently increases intracellular calcium concentration in cultured C2C12 myotubes. Thus our objective was to test our hypothesis that myotubes with increased levels of the HERG1 protein would have increased calpain activity. We transfected C2C12 myotubes with either an adenovirus encoding HERG1 or an appropriate control virus and, in both experimental groups, measured: 1) myotube size using ImageJ; 2) MuRF1 protein abundance using immunoblot; and 3) calpain activity in depolarized and non‐depolarized myotubes using a Calpain‐Glo Assay Kit (ProMega; Madison, WI). Indeed, our data reveal that, relative to controls, myotubes expressing HERG1 undergo atrophy, experiencing: 1) a 55% decrease in size; 2) an increased abundance of the UPP E3 ligase MuRF1; and 3) an 18% increase in calpain activity. We conclude that HERG1 increases proteolytic calpain activity in cultured myotubes. Support or Funding Information This research was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number NIH NIAMS 1R03AR053706‐01A2 to ALP. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The substituted amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy), is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic, however, promising outcomes for the therapeutic utility of MDMA for post-traumatic stress disorder and the long-time use of the dopaminergic and noradrenergic-directed amphetamines in treatment of attention-deficit hyperactivity disorder and narcolepsy increases the importance of understanding this phenomenon. Previously, we identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. Here, we performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. Our findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser.

Exposure of pancreatic β‐cells to glucose generates concomitant oscillations in Ca 2+ and cAMP which regulate insulin secretion, an essential function of β‐cells that promotes glucose homeostasis. Ca 2+ influx through the L‐type Ca 2+ channels Ca v 1.2 and Ca v 1.3 is further amplified by Ca 2+ released from the ER via the ryanodine receptor (RyR) through a process called Ca 2+ ‐induced Ca 2+ release (CICR). L‐type Ca 2+ channel blockers abolish glucose‐stimulated cAMP accumulation (GS‐cAMP); however, the role of CICR in this process is not well understood. We've demonstrated that expression of the Ca v 1.2 II‐III loop, a highly divergent region between the amino acid sequences of Ca v 1.2 and Ca v 1.3 that is required for critical protein‐protein interactions, displaces endogenous Ca v 1.2 channels from lipid raft domains in INS‐1 cells (Ca v 1.2/II‐III cells). Given our finding that Ca v 1.2 colocalizes with the ER Ca 2+ release channel RyR2 in INS‐1 cells, expression of the Ca v 1.2 II‐III loop may perturb proper spatial coupling of Ca v 1.2 with RyR. Consistent with this, we found that CICR is uncoupled from Ca v 1.2‐mediated Ca 2+ influx in Ca v 1.2/II‐III cells. To examine the role of CICR in GS‐cAMP, we expressed the Epac1‐based, cytosolic FRET sensor H187 in control INS‐1 and CICR‐deficient Ca v 1.2/II–III cells. When cAMP production was stimulated with the adenylyl cyclase activator forskolin, a robust response was detected in both control INS‐1 and Ca v 1.2/II–III cells, and there was no significant difference between them. In response to glucose (18 mM), we detected cAMP accumulation above baseline in control INS‐1 cells; however, glucose failed to elicit a detectable increase in Ca v 1.2/II–III cells. Further, both the L‐type Ca 2+ channel blocker nicardipine (2 μM) and RyR inhibitor ryanodine (20 μM) completely abolished GS‐cAMP in control INS‐1 cells, suggesting that Ca 2+ influx through L‐type Ca 2+ channels and CICR are required for GS‐cAMP. Use of a plasma membrane‐targeted, Epac2‐based FRET sensor revealed that in Ca v 1.2/II–III cells, L‐type Ca 2+ channel‐dependent GS‐cAMP is present at the plasma membrane. Taken together, Ca 2+ influx through L‐type Ca 2+ channels is sufficient to cause local GS‐cAMP; however, CICR largely amplifies the signal. Given our observation that GS‐cAMP is diminished in Ca v 1.2/II–III cells, we predicted that Ca v 1.2‐mediated CICR is preferentially coupled with GS‐cAMP. To test this, we utilized INS‐1 cells stably expressing dihydropyridine‐insensitive (DHPi) Ca v 1.2 and Ca v 1.3 channels. Here, the introduced DHPi channel is resistant to the DHP isradipine (2 μM); however, it remains sensitive to diltiazem (500 μM). As expected, we found that glucose markedly stimulated cAMP accumulation in both Ca v 1.2/DHPi and Ca v 1.3/DHPi cells. Surprisingly, isradipine significantly but incompletely inhibited GS‐cAMP in both cell lines, whereas diltiazem completely abolished the responses. Thus, Ca v 1.2 or Ca v 1.3 can sustain GS‐cAMP in the absence of the corresponding channel. Overall, we conclude that both Ca 2+ influx through L‐type Ca 2+ channels and CICR are required for efficient GS‐cAMP, and both Ca v 1.2 and Ca v 1.3 are involved in this process. Support or Funding Information This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Disease (R01 DK064736) (to Gregory Hockerman) and a Purdue Research Foundation Grant (to Evan Pratt and Gregory Hockerman).

Bimolecular fluorescence complementation (BiFC) is a fluorescence imaging technique used to visualize protein-protein interactions (PPIs) in live cells and animals. One unique application of BiFC is to reveal subcellular localization of PPIs. The superior signal-to-noise ratio of BiFC in comparison with fluorescence resonance energy transfer or bioluminescence resonance energy transfer enables its wide applications. Here, we describe how confocal microscopy can be used to detect and quantify PPIs and their subcellular localization. We use basic leucine zipper transcription factor proteins as an example to provide a step-by-step BiFC protocol using a Nikon A1 confocal microscope and NIS-Elements imaging software. The protocol given below can be readily adapted for use with other confocal microscopes or imaging software.