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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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Harvey et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 IP 3 Receptor Binding protein released with Inositol 1,4,5 Trisphosphate (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., (© 2022. The Author(s).)

Background: The potassium channel encoded by the ether-a-gogo-related gene 1A ( erg1 a) 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.

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Nifedipine and FPL 64176 (FPL), which block and potentiate L-type voltage-gated Ca 2+ 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 IC 50 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 IC 50 of nifedipine to 42 nM. FPL increased current amplitude with an EC 50 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 μ 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 (IC 50 ∼93 μ M), 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 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., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We previously reported that INS-1 cells expressing the intracellular II-III loop of the L-type Ca(2+) channel Cav1.2 (Cav1.2/II-III cells) are deficient in Ca(2+)-induced Ca(2+) release (CICR). Here we show that glucose-stimulated ERK 1/2 phosphorylation (GSEP) is slowed and reduced in Cav1.2/II-III cells compared to INS-1 cells. This parallels a decrease in glucose-stimulated cAMP accumulation (GS-cAMP) in Cav1.2/II-III cells. Influx of Ca(2+) via L-type Ca(2+) channels and CICR play roles in both GSEP and GS-cAMP in INS-1 cells since both are inhibited by nicardipine or ryanodine. Further, the Epac1-selective inhibitor CE3F4 abolishes glucose-stimulated ERK activation in INS-1 cells, as measured using the FRET-based sensor EKAR. The non-selective Epac antagonist ESI-09 but not the Epac2-selective antagonist ESI-05 nor the PKA antagonist Rp-cAMPs inhibits GSEP in both INS-1 and Cav1.2/II-III cells. We conclude that L-type Ca(2+) channel-dependent cAMP accumulation, that's amplified by CICR, activates Epac1 and drives GSEP in INS-1 cells., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

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.

Since the discovery of the GABA(B) agonist and muscle relaxant baclofen, there have been substantial advancements in the development of compounds that activate the GABA(B) receptor as agonists or positive allosteric modulators. For the agonists, most of the existing structure-activity data apply to understanding the role of substituents on the backbone of GABA as well as replacing the carboxylic acid and amine groups. In the cases of the positive allosteric modulators, the allosteric binding site(s) and structure-activity relationships are less well-defined; however, multiple classes of molecules have been discovered. The recent report of the X-ray structure of the GABA(B) receptor with bound agonists and antagonists provides new insights for the development of compounds that bind the orthosteric site of this receptor. From a therapeutic perspective, these data have enabled efforts in drug discovery in areas of addiction-related behavior, the treatment of anxiety, and the control of muscle contractility.

We investigated the role of Cav1.2 in pancreatic β-cell function by expressing a Cav1.2 II-III loop/green fluorescent protein fusion in INS-1 cells (Cav1.2/II-III cells) to disrupt channel-protein interactions. Neither block of KATP channels nor stimulation of membrane depolarization by tolbutamide was different in INS-1 cells compared with Cav1.2/II-III cells, but whole-cell Cav current density was significantly increased in Cav1.2/II-III cells. Tolbutamide (200 μM) stimulated insulin secretion and Ca(2+) transients in INS-1 cells, and Cav1.2/II-III cells were completely blocked by nicardipine (2 μM), but thapsigargin (1 μM) blocked tolbutamide-stimulated secretion and Ca(2+) transients only in INS-1 cells. Tolbutamide-stimulated endoplasmic reticulum [Ca(2+)] decrease was reduced in Cav1.2/II-III cells compared with INS-1 cells. However, Ca(2+) transients in both INS-1 cells and Cav1.2/II-III cells were significantly potentiated by 8-pCPT-2'-O-Me-cAMP (5 μM), FPL-64176 (0.5 μM), or replacement of extracellular Ca(2+) with Sr(2+). Glucose (10 mM) + glucagon-like peptide-1 (10 nM) stimulated discrete spikes in [Ca(2+)]i in the presence of verapamil at a higher frequency in INS-1 cells than in Cav1.2/II-II cells. Glucose (18 mM) stimulated more frequent action potentials in Cav1.2/II-III cells and primary rat β-cells expressing the Cav1.2/II-II loop than in control cells. Further, apamin (1 μM) increased glucose-stimulated action potential frequency in INS-1 cells, but not Cav1.2/II-III cells, suggesting that SK channels were not activated under these conditions in Cav1.2/II-III loop-expressing cells. We propose the II-III loop of Cav1.2 as a key molecular determinant that couples the channel to Ca(2+)-induced Ca(2+) release and activation of SK channels in pancreatic β-cells.

Skeletal muscle (SKM) atrophy is a potentially debilitating condition induced by muscle disuse, denervation, many disease states, and aging. The ubiquitin proteasome pathway (UPP) contributes greatly to the protein loss suffered in muscle atrophy. The MERG1a K(+) channel is known to induce UPP activity and atrophy in SKM. It has been further demonstrated that the mouse ether-a-gogo-related gene (Merg)1a channel modulates expression of MURF1, an E3 ligase component of the UPP, while it does not affect expression of the UPP E3 ligase Mafbx/ATROGIN1. Because the UBR2 E3 ligase is known to participate in SKM atrophy, we have investigated the effect of Merg1a expression and hind limb suspension on Ubr2 expression. Here, we report that hind limb suspension results in a significant 25.6% decrease in mouse gastrocnemius muscle fiber cross sectional area (CSA) and that electro-transfer of Merg1a alone into gastrocnemius muscles yields a 15.3% decrease in CSA after 7 days. More interestingly, we discovered that hind limb suspension caused a significant 8-fold increase in Merg1a expression and a significant 4.7-fold increase in Ubr2 transcript after 4 days, while electro-transfer of Merg1a into gastrocnemius muscles resulted in a significant 6.2-fold increase in Merg1a transcript after 4 days but had no effect on Ubr2 expression. In summary, the MERG1a K(+) channel, known to induce atrophy and MURF1 E3 ligase expression, does not affect UBR2 E3 ligase transcript levels. Therefore, to date, the MERG1a channel's contribution to UPP activity appears mainly to be through up-regulation of Murf1 gene expression.

Introduction: We investigated the mechanism by which the MERG1a K+ channel increases ubiquitin proteasome proteolysis (UPP)., Methods: Hindlimb suspension and electro-transfer of Merg1a cDNA into mouse gastrocnemius muscles induced atrophy., Results: Atrophic gastrocnemius muscles of hindlimb-suspended mice express Merg1a, Murf1, and Mafbx genes. Electrotransfer of Merg1a significantly decreases muscle fiber size (12.6%) and increases UPP E3 ligase Murf1 mRNA (2.1-fold) and protein (23.7%), but does not affect Mafbx E3 ligase expression. Neither Merg1a-induced decreased fiber size nor Merg1a-induced increased Murf1 expression is curtailed significantly by coexpression of inactive HR-Foxo3a, a gene encoding a transcription factor known to induce Mafbx expression., Conclusions: The MERG1a K+ channel significantly increases expression of Murf1, but not Mafbx. We explored this expression pattern by expressing inactive Foxo3a and showing that it is not involved in MERG1a-mediated expression of Murf1. These findings suggest that MERG1a may not modulate Murf1 expression through the AKT/FOXO pathway., (Copyright © 2013 Wiley Periodicals, Inc.)

Adenylyl cyclase (AC) isoforms are implicated in several physiologic processes and disease states, but advancements in the therapeutic targeting of AC isoforms have been limited by the lack of potent and isoform-selective small-molecule modulators. The discovery of AC isoform-selective small molecules is expected to facilitate the validation of AC isoforms as therapeutic targets and augment the study of AC isoform function in vivo. Identification of chemical probes for AC2 is particularly important because there are no published genetic deletion studies and few small-molecule modulators. The present report describes the development and implementation of an intact-cell, small-molecule screening approach and subsequent validation paradigm for the discovery of AC2 inhibitors. The NIH clinical collections I and II were screened for inhibitors of AC2 activity using PMA-stimulated cAMP accumulation as a functional readout. Active compounds were subsequently confirmed and validated as direct AC2 inhibitors using orthogonal and counterscreening assays. The screening effort identified SKF-83566 [8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrobromide] as a selective AC2 inhibitor with superior pharmacological properties for selective modulation of AC2 compared with currently available AC inhibitors. The utility of SKF-83566 as a small-molecule probe to study the function of endogenous ACs was demonstrated in C2C12 mouse skeletal muscle cells and human bronchial smooth muscle cells.

The design, synthesis, biological evaluation, and in vivo studies of difluoromethyl ketones as GABAB agonists that are not structurally analogous to known GABAB agonists, such as baclofen or 3-aminopropyl phosphinic acid, are presented. The difluoromethyl ketones were assembled in three synthetic steps using a trifluoroacetate-release aldol reaction. Following evaluation at clinically relevant GABA receptors, we have identified a difluoromethyl ketone that is a potent GABAB agonist, obtained its X-ray structure, and presented preliminary in vivo data in alcohol-preferring mice. The behavioral studies in mice demonstrated that this compound tended to reduce the acoustic startle response, which is consistent with an anxiolytic profile. Structure-activity investigations determined that replacing the fluorines of the difluoromethyl ketone with hydrogens resulted in an inactive analogue. Resolution of the individual enantiomers of the difluoromethyl ketone provided a compound with full biological activity at concentrations less than an order of magnitude greater than the pharmaceutical, baclofen.

Tolbutamide and gliclazide block the K(ATP) channel K(ir)6.2/Sur1, causing membrane depolarization and stimulating insulin secretion in pancreatic beta cells. We examined the ability of the EPAC-selective cAMP analog 8-pCPT-2'-O-Me-cAMP-AM to potentiate the action of these drugs and the mechanism that might account for it. Insulin secretion stimulated by both 200 μM tolbutamide and 20 μM gliclazide, concentrations that had equivalent effects on membrane potential, was inhibited by thapsigargin (1 μM) or the L-type Ca(2+) channel blocker nicardipine (2 μM) and was potentiated by 8-pCPT-2'-O-Me-cAMP-AM at concentrations ≥2 μM in INS-1 cells. Ca(2+) transients stimulated by either tolbutamide or gliclazide were inhibited by thapsigargin or nicardipine and were significantly potentiated by 8-pCPT-2'-O-Me-cAMP-AM at 5 μM but not 1 μM. Both tolbutamide and gliclazide stimulated phospholipase C activity; however, only gliclazide did so independently of its activity at K(ATP) channels, and this activity was partially inhibited by pertussis toxin. 8-pCPT-2'-O-Me-cAMP-AM alone (5 μM) did not stimulate insulin secretion, but did increase intracellular Ca(2+) concentration significantly, and this activity was inhibited by 25 μM 2-aminoethoxydiphenylborate (2-APB) or the removal of extracellular Ca(2+). 8-pCPT-2'-O-Me-cAMP-AM potentiation of insulin secretion stimulated by tolbutamide was markedly inhibited by 2-APB (25 μM) and enhanced by the PKC inhibitor bisindolylmaleimide I (1 μM). Our data demonstrate that the actions of both tolbutamide and gliclazide are strongly potentiated by 8-pCPT-2'-O-Me-cAMP-AM, that gliclazide can stimulate phospholipase C activity via a partially pertussis toxin-sensitive mechanism, and that 8-pCPT-2'-O-Me-cAMP-AM potentiation of tolbutamide action may involve activation of a 2-APB-sensitive Ca(2+) influx.

The binding site within the L-type Ca(2+) channel Ca(v)1.2 for neutral dihydropyridines is well characterized. However, the contributions of the alkylamino side chains of charged dihydropyridines such as amlodipine and nicardipine to channel block are not clear. We tested the hypothesis that the distinct locations of the charged side chains on amlodipine and nicardipine would confer distinct properties of channel block by these two drugs. Using whole-cell voltage clamp, we investigated block of wild type Ca(v) 2.1, wild type Ca(v)1.2, and Ca(v)1.2/Dihydropyridine insensitive, a mutant channel insensitive to neutral DHPs, by amlodipine and nicardipine. The potency of nicardipine and amlodipine for block of closed (stimulation frequency of 0.05 Hz) Ca(v)1.2 channels was not different (IC(50) values of 60 nM and 57 nM, respectively), but only nicardipine block was enhanced by increasing the stimulation frequency to 1 Hz. The frequency-dependent block of Ca(v)1.2 by nicardipine is the result of a strong interaction of nicardipine with the inactivated state of Ca(v)1.2. However, nicardipine block of Ca(v)1.2/Dihydropyridine insensitive was much more potent than block by amlodipine (IC(50) values of 2.0 μM and 26 μM, respectively). A mutant Ca(v)2.1 channel containing the neutral DHP binding site (Ca(v)2.1/Dihydropyridine sensitive) was more potently blocked by amlodipine (IC(50)=41 nM) and nicardipine (IC(50)=175 nM) than the parent Ca(v)2.1 channel. These data suggest that the alkylamino group of nicardipine and amlodipine project into distinct regions of Ca(v)1.2 such that the side chain of nicardipine, but not amlodipine, contributes to the potency of closed-channel block, and confers frequency-dependent block., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The incretin peptides, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), potentiate glucose-stimulated insulin secretion (GSIS) and beta-cell proliferation and differentiation. Ca(2+) influx via voltage-gated L-type Ca(2+) channels is required for GLP-1 and GIP potentiation of GSIS. We investigated the role of the L-type Ca(2+) channels Ca(v)1.2 and Ca(v)1.3 in mediating GLP-1- and GIP-stimulated events in INS-1 cells and INS-1 cell lines expressing dihydropyridine-insensitive (DHPi) mutants of either Ca(v)1.2 or Ca(v)1.3. Ca(v)1.3/DHPi channels supported full potentiation of GSIS by GLP-1 (50 nM) compared with untransfected INS-1 cells. However, GLP-1-potentiated GSIS mediated by Ca(v)1.2/DHPi channels was markedly reduced compared with untransfected INS-1 cells. In contrast, GIP (10 nM) potentiation of GSIS mediated by both Ca(v)1.2/DHPi and Ca(v)1.3/DHPi channels was similar to that observed in untransfected INS-1 cells. Disruption of intracellular Ca(2+) release with thapsigargin, ryanodine, or 2-aminoethyldiphenylborate and inhibition of protein kinase A (PKA) or protein kinase C (PKC) significantly reduced GLP-1 potentiation of GSIS by Ca(v)1.3/DHPi channels and by endogenous L-type channels in INS-1 cells, but not by Ca(v)1.2/DHPi channels. Inhibition of glucose-stimulated phospholipase C activity with 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) did not inhibit potentiation of GSIS by GLP-1 in INS-1 cells. In contrast, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and 2'-amino-3'-methoxyflavone (PD98059), an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase, both markedly inhibited GLP-1 potentiation of GSIS by endogenous channels in INS-1 cells and Ca(v)1.3/DHPi channels, but not by Ca(v)1.2/DHPi channels. Thus, Ca(v)1.3 is preferentially coupled to GLP-1 potentiation of GSIS in INS-1 cells via a mechanism that requires intact intracellular Ca(2+) stores, PKA and PKC activity, and activation of ERK1/2.

L-type Ca(2+) channels play a key role in the integration of physiological signals regulating insulin secretion that probably requires their localization to specific subdomains of the plasma membrane. We investigated the role of the intracellular II-III loop domains of the L-type channels Ca(v)1.2 and 1.3 in coupling of Ca(2+) influx with glucose-stimulated insulin secretion (GSIS) potentiated by the incretin hormone glucagon-like peptide (GLP)-1. In INS-1 cell lines expressing the Ca(v)1.2/II-III or Ca(v)1.3/II-III peptides, GLP-1 potentiation of GSIS was inhibited markedly, coincident with a decrease in GLP-1-stimulated cAMP accumulation and the redistribution of Ca(v)1.2 and Ca(v)1.3 out of lipid rafts. Neither the Ca(v)1.2/II-III nor the Ca(v)1.3/II-III peptide decreased L-type current density compared with untransfected INS-1 cells. GLP-1 potentiation of GSIS was restored by the L-type channel agonist 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL-64176). In contrast, potentiation of GSIS by 8-bromo-cAMP (8-Br-cAMP) was inhibited in Ca(v)1.2/II-III but not Ca(v)1.3/II-III cells. These differences may involve unique protein-protein interactions because the Ca(v)1.2/II-III peptide, but not the Ca(v)1.3/II-III peptide, immunoprecipitates Rab3-interacting molecule (RIM) 2 from INS-1 cell lysates. RIM2, and its binding partner Piccolo, localize to lipid rafts, and they may serve as anchors for Ca(v)1.2 localization to lipid rafts in INS-1 cells. These findings suggest that the II-III interdomain loops of Ca(v)1.2, and possibly Ca(v)1.3, direct these channels to membrane microdomains in which the proteins that mediate potentiation of GSIS by GLP-1 and 8-Br-cAMP assemble.

The Kv11.1 (also ERG1) K(+) channel underlies cardiac I(Kr), a current that contributes to repolarization in mammalian heart. In mice, I(Kr) current density decreases with development and studies suggest that changes in the structure and/or properties of the heteromultimeric I(Kr)/Kv11.1 channel are responsible. Here, using immunohistochemistry, we report that total Kv11.1 alpha subunit protein is more abundant in neonatal heart and is distributed throughout both adult and neonatal ventricles with greater abundance in epicardia. Immunoblots reveal that the alpha subunit alternative splice variant, Kv11.1a, is more abundant in adult heart while the Kv11.1b variant is more abundant in neonatal heart. Additionally, MinK channel subunit protein is shown to co-assemble with Kv11.1 protein and is more abundant in neonatal heart. In summary, Kv11.1/I(Kr) channel composition varies developmentally and the higher I(Kr) current density in neonatal heart is likely attributable to higher abundance of Kv11.1/I(Kr) channels, more specifically, the Kv11.1b splice variant.

Cardiac voltage-gated Ca2+ channels regulate the intracellular Ca2+ concentration and are therefore essential for muscle contraction, second messenger activation, gene expression and electrical signaling. As a first step in accessing the structural versus functional properties of the L-type Ca2+ channel in the heart, we have expressed a dihydropyridine (DHP)-insensitive CaV1.2 channel in rat ventricular myocytes and fibroblasts. Following isolation and culture, cells were infected with adenovirus expressing either LacZ or a mutant CaV1.2 channel (CaV1.2DHPi) containing the double mutation (T1039Y & Q1043M). This mutation renders the channel insensitive to neutral DHP compounds such as nisoldipine. The whole-cell, L-type Ca2+ current (ICa) measured in control myocytes was inhibited in a concentration-dependent manner by nisoldipine with an IC50 of 66 nM and complete block at 250 nM. In contrast, ICa in cells infected with AdCaV1.2DHPi was inhibited by only 35% by 500 nM nisoldipine but completely blocked by 50 microM diltiazem. In order to study CaV1.2DHPi in isolation, myocytes infected with AdCaV1.2DHPi were incubated with nisoldipine. Under this condition the cells expressed a large ICa (12 pA/pF) and displayed Ca2+ transients during field stimulation. Furthermore, addition of 2 microM forskolin and 100 microM 3-isobutyl-1-methylxanthine (IBMX), to stimulate protein kinase A, strongly increased IBa in the AdCaV1.2DHPi-infected cells. A Cd2+-sensitive IBa was also recorded in cardiac fibroblasts infected with AdCaV1.2DHPi. Thus, expression of CaV1.2DHPi will provide an important tool in studies of cardiac myocyte and fibroblast function.

Skeletal muscle atrophy results from an imbalance in protein degradation and protein synthesis and occurs in response to injury, various disease states, disuse, and normal aging. Current treatments for this debilitating condition are inadequate. More information about mechanisms involved in the onset and progression of muscle atrophy is necessary for development of more effective therapies. Here we show that expression of the mouse ether-a-go-go related gene (Merg1a) K+ channel is up-regulated in skeletal muscle of mice experiencing atrophy as a result of both malignant tumor expression and disuse. Further, ectopic expression of Merg1a in vivo induces atrophy in healthy wt-bearing mice, while expression of a dysfunctional Merg1a mutant suppresses atrophy in hindlimb-suspended mice. Treatment of hindlimb-suspended mice with astemizole, a known Merg1a channel blocker, inhibits atrophy in these animals. Importantly, in vivo expression of Merg1a in mouse skeletal muscle activates the ubiquitin proteasome pathway that is responsible for the majority of protein degradation that causes muscle atrophy, yet expression of a dysfunctional Merg1a mutant decreases levels of ubiquitin-proteasome proteolysis. Thus, expression of Merg1a likely initiates atrophy by activating ubiquitin-proteasome proteolysis. This gene and its product are potential targets for prevention and treatment of muscle atrophy.

Using insulin-secreting cell line (INS)-1 cells stably expressing dihydropyridine-insensitive mutants of either Cav1.2 or Cav1.3, we previously demonstrated that Cav1.3 is preferentially coupled to insulin secretion and [Ca2+]i oscillations stimulated by 11.2 mM glucose. Using the same system, we found that insulin secretion in 7.5 mM glucose plus 1 mM 8-bromo-cAMP (8-Br-cAMP) is mediated by both Cav1.2 and Cav1.3. Treatment of INS-1 cells or INS-1 cells stably expressing Cav1.2/dihydropyridine-insensitive (DHPi) channels in the presence of 10 microM nifedipine, with effector-specific cAMP analogs 8-(4-chlorophenylthio)-2'-O-methyladenosine-cAMP [8-pCPT-2'-O-Me-cAMP; 100 microM; Exchange Protein directly Activated by cAMP 2 (Epac2)-selective] or N6-benzoyl-cAMP [50 microM; Protein Kinase A (PKA)-selective] partially increased insulin secretion. Secretion stimulated by a combination of the two cAMP analogs was additive and comparable with that stimulated by 1 mM 8-Br-cAMP. In INS-1 cells stably expressing Cav1.3/DHPi in the presence of 10 microM nifedipine, N6-benzoyl-cAMP, but not 8-pCPT-2'-O-Me-cAMP, significantly increased glucose-stimulated insulin secretion. However, the combination of N6-benzoyl-cAMP and 8-pCPT-2'-O-Me-cAMP significantly increased glucose-stimulated secretion compared with N6-benzoyl-cAMP alone. In INS-1 cells, 8-Br-cAMP potentiation of insulin secretion in 7.5 mM glucose is blocked by thapsigargin (1 microM) and ryanodine (0.5 microM). In contrast, ryanodine has no effect on insulin secretion or [Ca2+]i oscillations stimulated by 11.2 mM glucose in INS-1 cells. Our data suggest that both Cav1.2 and Cav1.3 mediate insulin secretion stimulated by 7.5 mM glucose and cAMP via a mechanism that requires internal stores of Ca2+. Furthermore, cAMP modulation of secretion mediated by Cav1.2 seems to involve both Epac2 and PKA independently. In contrast, cAMP modulation of Cav1.3-mediated secretion depends upon PKA activation, whereas the contribution of Epac2 is dependent upon PKA activation.

SNARE-proteins (soluble NSF-attachment protein receptor) are important for Ca(2+)-dependent exocytosis. We have used capacitance measurements and confocal imaging to dissect the role of synaptosomal protein of 25 kDa (SNAP-25) and syntaxin 1 in rapid exocytosis in insulin-secreting pancreatic beta-cells. Following immunoneutralization of syntaxin 1 and SNAP-25, exocytosis was strongly reduced and associated with a marked reduction in the size of the readily releasable pool (RRP) by 65% and 86% in the presence of the anti-SNAP-25 and anti-syntaxin 1 antibodies respectively. The size of the immediately releasable pool (IRP), a subset of RRP in close association with the voltage-dependent Ca(2+)-channels, was reduced to an equal extent. The reduction in IRP correlated with slowed release kinetics and the time constant (tau) increased from a control value of 16 to 36 ms and 51 ms after inclusion of anti-SNAP-25 and anti-syntaxin 1 antibodies respectively in the pipette solution. We further show that SNAP-25 and syntaxin 1 aggregate in clusters along the plasma membrane. The size of these clusters was estimated to be approximately 300 nm and every beta-cell contained approximately 400 SNAP-25/syntaxin 1 clusters. Whereas the inhibitory action of the anti-syntaxin 1 antibody on exocytosis could be attributed almost entirely to suppression of the voltage-dependent Ca(2+)-current (-40%), the effect of the anti-SNAP-25 antibody was not mediated by decreased Ca(2+)-entry and is more likely due to a direct interference with the exocytotic machinery. Our data are consistent with the concept that both syntaxin 1 and SNAP-25 are required for rapid exocytosis in beta-cells.

Verapamil block of Ca(v)1.2 is frequency-dependent and potentiated by Ca(2+). We examined the molecular determinants of these characteristics using mutations that effect Ca(2+) interactions with Ca(v)1.2. Mutant and wild-type Ca(v)1.2 channels were transiently expressed in tsA 201 cells with beta(1b) and alpha(2)delta subunits. The four conserved glutamates that compose the Ca(2+) selectivity filter in Ca(v)1.2 were mutated to Gln (E363Q, E709Q, E1118Q, E1419Q) and the adjacent conserved threonine in each domain was mutated to Ala (T361A, T707A, T1116A, T1417A). The L-type-specific residues in the domain III pore region (F1117G) and the C-terminal tail (I1627A) were also mutated and assayed for block by verapamil using whole-cell voltage-clamp recordings in 10 mM Ba(2+) or 10 mM Ca(2+). In Ba(2+), none of the pore-region mutations reduced the fraction of current blocked by 30 microM verapamil at 0.05 Hz stimulation. However, all of the pore-region mutations abolished Ca(2+) potentiation of verapamil block at 0.05 Hz. The T1116A, F1117G, E1118Q, and E1419Q mutations all significantly reduced frequency-dependent verapamil block (1-Hz stimulation) in both Ba(2+) and Ca(2+). The I1627A mutation, which disrupts Ca(2+)-dependent inactivation, increased the fraction of closed channels blocked by 30 microM verapamil in Ba(2+) but did not affect frequency-dependent block in Ba(2+) or Ca(2+). Our data suggest that the pore region of domain III may contribute to a high affinity verapamil binding site accessed during 1-Hz stimulation and that Ca(2+) binding to multiple sites may be required for potentiation of verapamil block of closed channels.

Plant defensins are a family of small Cys-rich antifungal proteins that play important roles in plant defense against invading fungi. Structures of several plant defensins share a Cys-stabilized alpha/beta-motif. Structural determinants in plant defensins that govern their antifungal activity and the mechanisms by which they inhibit fungal growth remain unclear. Alfalfa (Medicago sativa) seed defensin, MsDef1, strongly inhibits the growth of Fusarium graminearum in vitro, and its antifungal activity is markedly reduced in the presence of Ca(2+). By contrast, MtDef2 from Medicago truncatula, which shares 65% amino acid sequence identity with MsDef1, lacks antifungal activity against F. graminearum. Characterization of the in vitro antifungal activity of the chimeras containing portions of the MsDef1 and MtDef2 proteins shows that the major determinants of antifungal activity reside in the carboxy-terminal region (amino acids 31-45) of MsDef1. We further define the active site by demonstrating that the Arg at position 38 of MsDef1 is critical for its antifungal activity. Furthermore, we have found for the first time, to our knowledge, that MsDef1 blocks the mammalian L-type Ca(2+) channel in a manner akin to a virally encoded and structurally unrelated antifungal toxin KP4 from Ustilago maydis, whereas structurally similar MtDef2 and the radish (Raphanus sativus) seed defensin Rs-AFP2 fail to block the L-type Ca(2+) channel. From these results, we speculate that the two unrelated antifungal proteins, KP4 and MsDef1, have evolutionarily converged upon the same molecular target, whereas the two structurally related antifungal plant defensins, MtDef2 and Rs-AFP2, have diverged to attack different targets in fungi.

The link between Ca(2+) influx through the L-type calcium channels Ca(v)1.2 or Ca(v)1.3 and glucose- or KCl-induced [Ca(2+)](i) mobilization in INS-1 cells was assessed using the calcium indicator indo-1. Cells responded to 18 mM glucose or 50 mM KCl stimulation with different patterns in [Ca(2+)](i) increases, although both were inhibited by 10 microM nifedipine. Although KCl elicited a prolonged elevation in [Ca(2+)](i), glucose triggered oscillations in [Ca(2+)](i.) Ca(v)1.2/dihydropyridine-insensitive (DHPi) cells and Ca(v)1.3/DHPi cells, and stable INS-1 cell lines expressing either DHP-insensitive Ca(v)1.2 or Ca(v)1.3 channels showed normal responses to glucose. However, in 10 microM nifedipine, only Ca(v)1.3/DHPi cells maintained glucose-induced [Ca(2+)](i) oscillation. In contrast, both cell lines exhibited DHP-resistant [Ca(2+)](i) increases in response to KCl. The percentage of cells responding to glucose was not significantly decreased by nifedipine in Ca(v)1.3/DHPi cells but was greatly reduced in Ca(v)1.2/DHPi cells. In 10 microM nifedipine, KCl-elicited [Ca(2+)](i) elevation was retained in both Ca(v)1.2/DHPi and Ca(v)1.3/DHPi cells. In INS-1 cells expressing the intracellular II-III loop of Ca(v)1.3, glucose failed to elicit [Ca(2+)](i) changes, whereas INS-1 cells expressing the Ca(v)1.2 II-III loop responded to glucose with normal [Ca(2+)](i) oscillation. INS-1 cells expressing Ca(v)1.2/DHPi containing the II-III loop of Ca(v)1.3 demonstrated a nifedipine-resistant slow increase in [Ca(2+)](i) and nifedipine-resistant insulin secretion in response to glucose that was partially inhibited by diltiazem. Thus, whereas the II-III loop of Ca(v)1.3 may be involved in coupling Ca(2+) influx to insulin secretion, distinct structural domains are required to mediate the preferential coupling of Ca(v)1.3 to glucose-induced [Ca(2+)](i) oscillation.

Submicron-sized Au particles and Au/SiO(2) nanocomposites (superparticles) as large as 670 nm have been introduced into tsA201 cells with minimal cell trauma by cationic transfection systems. Successful implantations can be characterized by the expression of co-transfected DNA.

Diltiazem block of Cav1.2 is frequency-dependent and potentiated by Ca2+. We examined the molecular determinants of these characteristics using mutations that affect Ca2+ interactions with Cav1.2. Mutant and wild-type (WT) Cav1.2 channels were transiently expressed in tsA 201 cells with beta1b and alpha2delta subunits. The four conserved glutamates that compose the Ca2+ selectivity filter in Cav1.2 were mutated to Gln (E363Q, E709Q, E1118Q, E1419Q), and each single mutant was assayed for block by diltiazem using whole-cell voltage-clamp recordings in either 10 mM Ba2+ or 10 mM Ca2+. In Ba2+, none of the mutations affected the potency of diltiazem block of closed channels (0.05 Hz stimulation). However, frequency-dependent block (1Hz stimulation) was eliminated in the mutant E1419Q (domain IV), which recovered more rapidly than WT channels from inactivated channel block. Potentiation of diltiazem block of closed Cav1.2 channels in Ca2+ was abolished in the E1118Q, F1117G (domain III), and E1419Q mutants. Frequency-dependent block in Ca2+ was reduced compared with WT Cav1.2 in the F1117G, E1118Q, and E1419Q mutants. The C-terminal tail IQ domain mutation I1627A, which disrupts Ca2+ dependent inactivation, enhanced diltiazem block of closed channels in Ba2+. We conclude that, in Ba2+, E1419 slows recovery from diltiazem block of depolarized Cav1.2 channels, but in Ca2+, E1118, E1419, and F1117 form a Ca2+ binding site that mediates the potentiation of diltiazem block of both closed and inactivated Cav1.2 channels. Furthermore, Ca2+-dependent inactivation, which is impaired in E709Q, E1118Q, E1419Q, and I1627A, is not required for Ca2+ potentiation of diltiazem block.

L-Type Ca(2+) channel blockers inhibit glucose and KCl-stimulated insulin secretion by pancreatic beta cells. However, the role of the two distinct L-type channels expressed by beta cells, Ca(v)1.2 and Ca(v)1.3, in this process is not clear. Therefore, we stably transfected INS-1 cells with two mutant channel constructs, Ca(v)1.2DHPi or Ca(v)1.3 DHPi. Whole-cell patch-clamp recordings demonstrated that both mutant channels are insensitive to dihydropyridines (DHPs), but are blocked by diltiazem. INS-1 cells expressing Ca(v)1.3/DHPi maintained glucose- and KCl-stimulated insulin secretion in the presence of DHPs, whereas cells expressing Ca(v)1.2/DHPi demonstrated DHP resistance to only KCl-induced secretion. INS-1 cells were also stably transfected with cDNAs encoding the intracellular loop between domains II and III of either Ca(v)1.2 or Ca(v)1.3 (Ca(v)1.2/II-III or Ca(v)1.3/II-III). Glucose- and KCl-stimulated insulin secretion in Ca(v)1.2/II-III cells were not different from untransfected INS-1 cells. However, glucose-stimulated insulin secretion was completely inhibited and KCl-stimulated secretion was substantially resistant to inhibition by DHPs, but sensitive to omega-agatoxin IVA in Ca(v)1.3/II-III cells. Moreover, the L-type channel agonist FPL 64176 markedly enhanced KCl-stimulated secretion by Ca(v)1.3/II-III cells. Together, our results suggest that Ca(2+) influx via Ca(v)1.3 is preferentially coupled to glucose-stimulated insulin secretion in INS-1 cells.

KP4 is a virally encoded fungal toxin secreted by the P4 killer strain of Ustilago maydis. Previous studies demonstrated that this toxin inhibits growth of the target fungal cells by blocking calcium uptake rather than forming channels, as had been suggested previously. Unexpectedly, this toxin was also shown to inhibit voltage-gated calcium channel activity in mammalian cells. We used whole-cell patch-clamp techniques to further characterize this activity against mammalian cells. KP4 is shown to specifically block L-type calcium channels with weak voltage dependence to the block. Because KP4 activity is abrogated by calcium, KP4 probably binds competitively with calcium to the channel exterior. Finally, it is shown that chemical reagents that modify lysine residues reduce KP4 activity in both patch-clamp experiments on mammalian cells and in fungal killing assays. Because the only lysine residue is K42, this residue seems to be crucial for both mammalian and fungal channel activity. Our results defining the type of mammalian channel affected by this fungal toxin further support our contention that KP4 inhibits fungal growth by blocking transmembrane calcium flux through fungal calcium channels, and imply a high degree of structural homology between these fungal and mammalian calcium channels.

The benzothiazepine diltiazem blocks ionic current through L-type Ca(2+) channels, as do the dihydropyridines (DHPs) and phenylalkylamines (PAs), but it has unique properties that distinguish it from these other drug classes. Wild-type L-type channels containing alpha(1CII) subunits, wild-type P/Q-type channels containing alpha(1A) subunits, and mutants of both channel types were transiently expressed in tsA-201 cells with beta(1B) and alpha(2)delta subunits. Whole-cell, voltage-clamp recordings showed that diltiazem blocks L-type Ca(2+) channels approximately 5-fold more potently than it does P/Q-type channels. Diltiazem blocked a mutant P/Q-type channel containing nine amino acid changes that made it highly sensitive to DHPs, with the same potency as L-type channels. Thus, amino acids specific to the L-type channel that confer DHP sensitivity in an alpha(1A) background also increase sensitivity to diltiazem. Analysis of single amino acid mutations in domains IIIS6 and IVS6 of alpha(1CII) subunits confirmed the role of these L-type-specific amino acid residues in diltiazem block, and also indicated that Y1152 of alpha(1CII), an amino acid critical to both DHP and PA block, does not play a role in diltiazem block. Furthermore, T1039 and Y1043 in domain IIIS5, which are both critical for DHP block, are not involved in block by diltiazem. Conversely, three amino acid residues (I1150, M1160, and I1460) contribute to diltiazem block but have not been shown to affect DHP or PA block. Thus, binding of diltiazem to L-type Ca(2+) channels requires residues that overlap those that are critical for DHP and PA block as well as residues unique to diltiazem.

The activity of L-type Ca2+ channels is increased by dihydropyridine (DHP) agonists and inhibited by DHP antagonists, which are widely used in the therapy of cardiovascular disease. These drugs bind to the pore-forming alpha1 subunits of L-type Ca2+ channels. To define the minimal requirements for DHP binding and action, we constructed a high-affinity DHP receptor site by substituting a total of nine amino acid residues from DHP-sensitive L-type alpha1 subunits into the S5 and S6 transmembrane segments of domain III and the S6 transmembrane segment of domain IV of the DHP-insensitive P/Q-type alpha1A subunit. The resulting chimeric alpha1A/DHPS subunit bound DHP antagonists with high affinity in radioligand binding assays and was inhibited by DHP antagonists with high affinity in voltage clamp experiments. Substitution of these nine amino acid residues yielded 86% of the binding energy of the L-type alpha1C subunit and 92% of the binding energy of the L-type alpha1S subunit for the high-affinity DHP antagonist PN200-110. The activity of chimeric Ca2+ channels containing alpha1A/DHPS was increased 3.5 +/- 0.7-fold by the DHP agonist (-)Bay K8644. The effect of this agonist was stereoselective as in L-type Ca2+ channels since (+) Bay K8644 inhibited the activity of alpha1A/DHPS. The results show conclusively that DHP agonists and antagonists bind to a single receptor site at which they have opposite effects on Ca2+ channel activity. This site contains essential components from both domains III and IV, consistent with a domain interface model for binding and allosteric modulation of Ca2+ channel activity by DHPs.

Recent studies of the phenylalkylamine binding site in the alpha1C subunit of L-type Ca2+ channels have revealed three amino acid residues in transmembrane segment IVS6 that are critical for high affinity block and are unique to L-type channels. We have extended this analysis of the phenylalkylamine binding site to amino acid residues in transmembrane segment IIIS6 and the pore region. Twenty-two consecutive amino acid residues in segment IIIS6 were mutated to alanine and the conserved Glu residues in the pore region of each homologous domain were mutated to Gln. Mutant channels were expressed in tsA-201 cells along with the beta1b and alpha2delta auxiliary subunits. Assay for block of Ba2+ current by (-)-D888 at -60 mV revealed that mutation of five amino acid residues in segment IIIS6 and the pore region that are conserved between L-type and non-L-type channels (Tyr1152, Phe1164, Val1165, Glu1118, and Glu1419) and one L-type-specific amino acid (Ile1153) decreased affinity for (-)-D888 from 10-20-fold. Combination of the four mutations in segment IIIS6 increased the IC50 for block by (-)-D888 to approximately 9 microM, similar to the affinity of non-L-type Ca2+ channels for this drug. These results indicate that there are important determinants of phenylalkylamine binding in both the S6 segments and the pore regions of domains III and IV, some of which are conserved across the different classes of voltage-gated Ca2+ channels. A model of the phenylalkylamine receptor site at the interface between domains III and IV of the alpha1 subunit is presented.

The dihydropyridine Ca2+ antagonist drugs used in the therapy of cardiovacular disorders inhibit L-type Ca2+ channels by binding to a single high affinity site. Photoaffinity labeling and analysis of mutant Ca2+ channels implicate the IIIS6 and IVS6 segments in high affinity binding. The amino acid residues that are required for high affinity binding of dihydropyridine Ca2+ channel antagonists were probed by alanine-scanning mutagenesis of the alpha1C subunit, transient expression in mammalian cells, and analysis by measurements of ligand binding and block of Ba2+ currents through expressed Ca2+ channels. Eleven amino acid residues in transmembrane segments IIIS6 and IVS6 were identified whose mutation reduced the affinity for the Ca2+ antagonist PN200-110 by 2-25-fold. Both amino acid residues conserved among Ca2+ channels and those specific to L-type Ca2+ channels were found to be required for high affinity dihydropyridine binding. In addition, mutation F1462A increased the affinity for the dihydropyridine Ca2+ antagonist PN200-110 by 416-fold with no effect on the affinity for the Ca2+ agonist Bay K8644. The residues in transmembrane segments IIIS6 and IVS6 that are required for high affinity binding are primarily aligned on single faces of these two alpha helices, supporting a "domain interface model" of dihydropyridine binding and action in which the IIIS6 and IVS6 interact to form a high affinity dihydropyridine receptor site on L-type Ca2+ channels.

Synaptic transmission is regulated by G protein-coupled receptors whose activation releases G protein betagamma subunits that modulate presynaptic Ca2+ channels. The sequence motif QXXER has been proposed to be involved in the interaction between G protein betagamma subunits and target proteins including adenylyl cyclase 2. This motif is present in the intracellular loop connecting domains I and II (L I-II) of Ca2+ channel alpha1A subunits, which are modulated by G proteins, but not in alpha1C subunits, which are not modulated. Peptides containing the QXXER motif from adenylate cyclase 2 or from alpha1A block G protein modulation but a mutant peptide containing the sequence AXXAA does not, suggesting that the QXXER-containing peptide from alpha1A can competitively inhibit Gbetagamma modulation. Conversion of the R in the QQIER sequence of alpha1A to E as in alpha1C slows channel inactivation and shifts the voltage dependence of steady-state inactivation to more positive membrane potentials. Conversion of the final E in the QQLEE sequence of alpha1C to R has opposite effects on voltage-dependent inactivation, although the changes are not as large as those for alpha1A. Mutation of the QQIER sequence in alpha1A to QQIEE enhanced G protein modulation, and mutation to QQLEE as in alpha1C greatly reduced G protein modulation and increased the rate of reversal of G protein effects. These results indicate that the QXXER motif in L I-II is an important determinant of both voltage-dependent inactivation and G protein modulation, and that the amino acid in the third position of this motif has an unexpectedly large influence on modulation by Gbetagamma. Overlap of this motif with the consensus sequence for binding of Ca2+ channel beta subunits suggests that this region of L I-II is important for three different modulatory influences on Ca2+ channel activity.

Ca2+ influx through skeletal muscle Ca2+ channels and the force of contraction are increased in response to beta-adrenergic stimulation and high-frequency electrical stimulation. These effects are thought to be mediated by cAMP-dependent phosphorylation of the skeletal muscle Ca2+ channel. Modulation of the cloned skeletal muscle Ca2+ channel by cAMP-dependent phosphorylation and by depolarizing prepulses was reconstituted by transient expression in tsA-201 cells and compared to modulation of the native skeletal muscle Ca2+ channel as expressed in mouse 129CB3 skeletal muscle cells. The heterologously expressed Ca2+ channel consisting of alpha1, alpha2delta, and beta subunits gave currents that were similar in time course, current density, and dihydropyridine sensitivity to the native Ca2+ channel. cAMP-dependent protein kinase (PKA) stimulation by Sp-5,6-DCl-cBIMPS (cBIMPS) increased currents through both native and expressed channels two- to fourfold. Tail currents after depolarizations to potentials between -20 and +80 mV increased in amplitude and decayed more slowly as either the duration or potential of the depolarization was increased. The time- and voltage-dependent slowing of channel deactivation required the activity of PKA, because it was enhanced by cBIMPS and reduced or eliminated by the peptide PKA inhibitor PKI (5-24) amide. This voltage-dependent modulation of the cloned skeletal muscle Ca2+ channel by PKA also required anchoring of PKA by A-Kinase Anchoring Proteins because it was blocked by peptide Ht 31, which disrupts such anchoring. The results show that the skeletal muscle Ca2+ channel expressed in heterologous cells is modulated by PKA at rest and during depolarization and that this modulation requires anchored protein kinase, as it does in native skeletal muscle cells.

The crucial role of L-type Ca2+ channels in the initiation of cardiac and smooth muscle contraction has made them major therapeutic targets for the treatment of cardiovascular disease. L-type channels share a common pharmacological profile, including high-affinity voltage- and frequency-dependent block by the phenylalkylamines, the benz(othi)azepines, and the dihydropyridines. These drugs are thought to bind to three separate receptor sites on L-type Ca2+ channels that are allosterically linked. Results from different experimental approaches implicate the IIIS5, IIIS6, and IVS6 transmembrane segments of the alpha 1 subunits of L-type Ca2+ channels in binding of all three classes of drugs. Site-directed mutagenesis has identified single amino acid residues within the IIIS5, IIIS6, and IVS6 transmembrane segments that are required for high-affinity binding of phenylalkylamines and/or dihydropyridines, providing further support for identification of these transmembrane segments as critical elements of the receptor sites for these two classes of drugs. The close proximity of the receptor sites for phenylalkylamines, benz(othi)azepines, and dihydropyridines raises the possibility that individual amino acid residues may be required for high-affinity binding of more than one of these ligands. Therefore, we suggest that phenylalkylamines and dihydropyridines bind to different faces of the IIIS6 and IVS6 transmembrane segments and, in some cases, bind to opposite sides of the side chains of the same amino acid residues. The results support the domain interface model for binding and channel modulation by these three classes of drugs.

The phenylalkylamines (-)-D888, verapamil, and D600, cause voltage- and use-dependent block of L-type Ca2+ channels and differ from each other only in the number of methoxy groups on each of their two terminal phenyl rings. To study the effects of mutations in the phenylalkylamine receptor site on block by these drugs, wild-type and mutant Ca2+ channels were transiently expressed in the tsA-201 clone of human embryonic kidney 293 cells. The combined mutations Y1463A, A1467S, and I1470A (mutant YAI) in transmembrane segment S6 of domain IV of the alpha 1c subunit disrupted block by all three phenylalkylamines. Surprisingly, although this mutation reduced both resting block at -60 mV and depolarized block at +10 mV by (-)-D888, resting and depolarized block by verapamil and D600 were relatively unaffected. In contrast, for all three drugs, use-dependent block during repetitive stimulations was sharply reduced, and the rate of recovery from depolarized block was accelerated for YAI channels. Thus, the effects of the YAI mutation on apparent affinity were specific to (-)-D888, whereas effects on the kinetics of block were observed for all three drugs. Additional experiments with substitution of phenylalanine for Y1463 suggested that (-)-D888 affinity is specifically sensitive to removal of the hydroxyl group of Y1463, whereas effects on the kinetics of block by all three phenylalkylamines require larger molecular changes, perhaps related to residue size and hydrophobicity. Analysis of the data using a state-dependent model of drug block suggests that these kinetic differences are caused by both changes in drug access to the receptor site and affinity for binding to the inactivated state of the channel. The different effects of the YAI mutations on the actions of (-)-D888, verapamil, and D600 indicate that these residues interact differently with these closely related drugs.

The interactions between beta-adrenergic receptor (beta AR) antagonists and the beta(2)AR were studied with the use of photoaffinity labels. A proteolytic map of the receptor was made and confirmed through amino-terminal amino acid sequencing by locating sites of derivatization. [125I]Iodoazidothiophenylalprenolol (IAPTA) is a photoaffinity derivative of the beta AR antagonist alprenolol with a photoactivatable group on the aryloxy end of the molecule. IAPTA exclusively derivatizes a peptide consisting of transmembrane domains (TMs) 6 and 7 of the hamster lung beta(2)AR, supporting the contention that TMs 6 and 7 interact with the aryloxy portion of the beta AR antagonist pharmacophore. The beta AR antagonist photoaffinity labels [125I]iodoazidobenzylpindolol (IABP), [125I]iodoazidophenyl CGP-12177A (IAPCGP), and [125I]iodocyanopindololdiazarene (ICYPdz) are similar in that their photoactive moieties are attached to the amino end of the antagonist pharmacophore. IABP derivatized TMs 5-7 and a peptide containing TM 1 to approximately equal extents. IAPCGP derivatized Tms 6 and 7 >> TM 5 = TM 4 = TMs 2 and 3 = TM 1. ICYPdz derivatized TM 1 >> TMs 6 and 7 > Tm 4. We conclude that the aryloxy end of the beta AR antagonist pharmacophore is highly constrained within TMs 6 and 7, whereas the amino terminus is much less constrained and able to assume multiple conformations. Molecular dynamics simulations predict that IABP, IAPCGP, and ICYPdz favor a folded conformation, with both ends close together. Derivatization of TMs 6 and 7 by IABP, IAPCGP, and ICYPdz suggests the folded conformation of these compounds in the ligand binding pocket.

The high affinity phenylalkylamine (-)D888 blocks ion currents through L-type Ca2+ channels containing the alpha 1C subunit with an apparent Kd of 50 nM, but N-type Ca2+ channels in the pheochromocytoma cell line PC12 are blocked with a 100-fold higher Kd value of 5 microM. L-type Ca2+ channels containing alpha 1C subunits with the site-directed mutations Y1463A, A1467S, or I1470A in the putative transmembrane segment S6 in domain IV (IVS6) were 6-12 times less sensitive to block by (-)D888 than control alpha 1C. Ca2+ channels containing paired combinations of these mutations were even less sensitive to block by (-)D888 than the single mutants, and channels containing all three mutations were > 100 times less sensitive to (-)D888 block, similar to N-type Ca2+ channels. In addition, the Y1463A mutant and all combination mutants including the Y1463A mutation had altered ion selectivity, suggesting that Tyr-1463 faces the pore and is involved in ion permeation. Since these three critical amino acid residues are aligned on the same face of the putative IVS6 alpha-helix, we propose that they contribute to a receptor site in the pore that confers a high affinity block of L-type channels by (-)D888.