Gregory H. Hockerman

Differential Antifungal and Calcium Channel-Blocking Activity among Structurally Related Plant Defensins

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 α/β-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 Ca2+. 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 Ca2+ 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 Ca2+ 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.

Development of a High-Throughput Screening Paradigm for the Discovery of Small-Molecule Modulators of Adenylyl Cyclase: Identification of an Adenylyl Cyclase 2 Inhibitor

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.

Merg1a K+ channel induces skeletal muscle atrophy by activating the ubiquitin proteasome pathway

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 (Mergla) K+ channel is up‐regulated in skeletal muscle of mice experiencing atrophy as a result of both malignant tumor expression and disuse. Further, ec‐topic expression of Mergla in vivo induces atrophy in healthy wt‐bearing mice, while expression of a dysfunctional Mergla mutant suppresses atrophy in hindlimb‐suspended mice. Treatment of hindlimb‐suspended mice with astemizole, a known Mergla channel blocker, inhibits atrophy in these animals. Importantly, in vivo expression of Mergla 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 Mergla mutant decreases levels of ubiquitin‐proteasome proteolysis. Thus, expression of Mergla likely initiates atrophy by activating ubiquitin‐proteasome proteolysis. This gene and its product are potential targets for prevention and treatment of muscle atrophy.—Wang, X., Hockerman, G. H., Green, H. W. III, Babbs, C. F., Mohammad, S. I., Gerrard, D., Latour, M. A., London, B., Hannon, K. M., Pond, A. L. Mergla K+ channel induces skeletal muscle atrophy by activating the ubiquitin proteasome pathway. FASEB J. 20, E803–E811 (2006)

Kv11.1 Channel Subunit Composition Includes MinK and Varies Developmentally in Mouse Cardiac Muscle

The Kv11.1 (also ERG1) K+ channel underlies cardiac IKr, a current that contributes to repolarization in mammalian heart. In mice, IKr current density decreases with development and studies suggest that changes in the structure and/or properties of the heteromultimeric IKr/Kv11.1 channel are responsible. Here, using immunohistochemistry, we report that total Kv11.1 α 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 α 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/IKr channel composition varies developmentally and the higher IKr current density in neonatal heart is likely attributable to higher abundance of Kv11.1/IKr channels, more specifically, the Kv11.1b splice variant. Developmental Dynamics 237:2430–2437, 2008.

Potentiation of Sulfonylurea Action by an EPAC-selective cAMP Analog in INS-1 Cells: Comparison of Tolbutamide and Gliclazide, and a Potential Role for EPAC Activation of a 2-APB-sensitive Ca2+ Influx.

Tolbutamide and gliclazide block the KATP channel Kir6.2/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 Ca2+ channel blocker nicardipine (2 μM) and was potentiated by 8-pCPT-2′-O-Me-cAMP-AM at concentrations ≥2 μM in INS-1 cells. Ca2+ 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 KATP 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 Ca2+ concentration significantly, and this activity was inhibited by 25 μM 2-aminoethoxydiphenylborate (2-APB) or the removal of extracellular Ca2+. 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 Ca2+ influx.

Nanoprobe implantation into mammalian cells by cationic transfection

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.

Distinct properties of amlodipine and nicardipine block of the voltage-dependent Ca2+ channels Cav1.2 and Cav2.1 and the mutant channels Cav1.2/Dihydropyridine insensitive and Cav2.1/Dihydropyridine sensitive

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.

Ca2+ influx through L-type Ca2+ channels and Ca2+-induced Ca2+ release regulate cAMP accumulation and Epac1-dependent ERK 1/2 activation in INS-1 cells

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, thats amplified by CICR, activates Epac1 and drives GSEP in INS-1 cells.

Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells.

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.

The mERG1a channel modulates skeletal muscle MuRF1, but not MAFbx, 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. Muscle Nerve 49:378–388, 2014