Mesenchymal stem/stromal cells engineered to express the protease inhibitor alpha-1 antitrypsin for the treatment of inflammatory lung diseases

Chronic inflammatory diseases are a growing global health problem. Chronic obstructive pulmonary disease (COPD) represents a major cause of morbidity and mortality, and is currently the third leading cause of death worldwide. COPD is a devastating progressive lung disease characterized by airflow limitation, chronic bronchitis and bronchiolitis, emphysema, and small airway remodeling. There are no curative options for COPD to date, thus there is a desperate need for novel approaches and treatments. To this end, the goal of this thesis work was to develop and evaluate a novel cell product composed of mesenchymal stem/stromal cells (MSCs) genetically modified to express the protease inhibitor alpha-1 antitrypsin (AAT), thus combining the potent immunomodulatory and cytoprotective properties of MSCs, and of AAT. A major focus was the development of an optimized expression construct/vector system for MSC engineering. Eight versions of gamma-retroviral-based vectors were designed and tested for optimal transgenic expression of human AAT and puromycin-N-acetyltransferase (pac) for antibiotic selection. The constructs differed in the gene promoter used for driving AAT expression, and in the relative position of the expression cassettes within the vector backbone. The vectors were then compared with regards to viral titers, transduction efficacy of primary human MSCs, AAT expression in the supernatant of transduced cells, and cell yield after antibiotic selection. The best results for all parameters were obtained when AAT was expressed from the human eukaryotic translation elongation factor 1 alpha 1 short (EFS) promoter, and the pac gene was linked via an internal ribosome entry site (IRES) sequence. This lead candidate expression cassette was then transferred to a lentiviral backbone to assess if transduction and AAT expression from MSCs could be further increased by using this viral system. While values were higher with the gamma-retroviral vector at low multiplicities of infection (MOIs), transduction reached a plateau and no further increase was possible even when higher MOIs were used. Transduction with the lentiviral construct, however, followed a clear dose-response, reaching transduction efficacies >80% at the highest MOI tested. In vitro functionality of the protease inhibitor AAT expressed from MSCs was confirmed using a neutrophil elastase inhibition assay. A process for large scale transduction and expansion of gene-modified MSCs was subsequently developed. To evaluate the potential therapeutic benefit of AAT-MSCs in vivo, a mouse model of elastase-induced emphysema was used. Treatment of the mice with AAT-MSCs resulted in significant improvement of pulmonary function parameters, while only a slight functional amelioration was observed after treatment with non-modified MSCs. Histopathologic examination of AAT-MSC-treated mice revealed a significant decrease of airspace enlargement, indicating regeneration of pulmonary tissue. Again, the effect was smaller after treatment with non-modified MSCs, demonstrating a more than additive effect of combining AAT and MSCs. In conclusion, the present study provides the first in vivo proof of concept for the treatment of emphysematous COPD using engineered AAT-MSCs.Chronisch entzündliche Erkrankungen sind heutzutage ein globales, im Steigen begriffenes Gesundheitsproblem. Ein wichtiges Beispiel ist die chronisch obstruktive Lungenerkrankung (COPD), eine verheerende, fortschreitende Erkrankung der Lunge mit hoher Morbidität und Mortalität, gegenwärtig die dritthäufigste Todesursache weltweit. Symptome der COPD sind Atemnot, eine chronische Entzündung der Bronchien und Bronchiolen, die Entwicklung eines Emphysems und ein Umbau der engen Atemwege. Da bislang eine Heilung der COPD nicht möglich ist, besteht ein dringender Bedarf an innovativen Therapieansätzen. Wir entwickelten ein neuartiges Zellprodukt, basierend auf mesenchymalen Stamm-/Stromazellen (MSCs), die genetisch modifiziert wurden, sodass sie den Protease-Inhibitor Alpha-1 Antitrypsin (AAT) exprimieren. Diese Kombination ermöglicht es, die immunmodulatorischen und zytoprotektiven Eigenschaften der MSCs mit jenen von AAT in einem Produkt zu vereinen. Dabei wurde in der vorliegenden Arbeit ein Fokus auf die Entwicklung eines optimierten Expressionskonstruktes gelegt. Es wurden acht gamma-retrovirale Vektoren für die Expression von AAT und Puromycin-N-Acetyltransferase (pac), das eine Antibiotikaselektion genmodifizierter Zellen erlaubt, konzipiert und kloniert. Diese Konstrukte unterscheiden sich sowohl im Promoter, der für die AAT-Expression verwendet wird, als auch in der relativen Anordnung der Expressionskassetten im Plasmid. Die Auswahl des besten Konstrukts wurde anhand eines Vergleichs der Vektoren hinsichtlich der erzielten viralen Titer, der Transduktionseffizienz von primären humanen MSCs, des AAT-Gehalts im Zellkulturüberstand von transduzierten MSCs sowie der Zellausbeute nach Antibiotikaselektion durchgeführt. Die jeweils besten Ergebnisse wurden erzielt, wenn AAT von der kurzen Form des Promoters des humanen eukaryotischen Translationselongationsfaktors 1 alpha 1 (EFS) exprimiert wurde, und das pac Gen durch eine interne ribosomale Eintrittsstelle- (IRES)-Sequenz verknüpft war. Diese favorisierte Expressionskassette wurde in ein lentivirales Plasmid transferiert, um zu untersuchen, ob Transduktion und AAT-Expression in einem solchen System weiter verbessert werden können. Es zeigte sich, dass mittels des gamma-retroviralen Vektors größere Transduktionseffizienzen und AAT-Mengen bei niedriger Multiplizität der Infektion (MOI) erzielt werden konnten, aber bei höheren MOIs keine zusätzliche Verbesserung zu erreichen war. Im Gegensatz dazu folgten Transduktion und AAT-Expression mittels des lentiviralen Vektors einer eindeutigen Dosis-Wirkung, die bei hohen MOIs in Transduktionseffizienzen von >80% resultierte. Ein Neutrophile Elastase Inhibitionstest bestätigte die in vitro Funktionalität von MSC-sezerniertem AAT als Proteaseinhibitor, bevor Transduktions- und Expansionsprozesse zur Herstellung von AAT-MSCs im Großformat etabliert wurden. Um die therapeutische Wirkung von AAT-MSCs in vivo zu untersuchen, wurde ein Mausmodell eines Elastase-induzierten Emphysems durchgeführt. Es zeigte sich, dass die Behandlung von Mäusen mit AAT-MSCs zu einer signifikanten Verbesserung der Lungenfunktion der Tiere führte, während nicht-modifizierte MSCs nur eine geringfügige Wiederherstellung der pulmonalen Funktion bewirkten. Die histopathologische Analyse ergab, dass die Überblähung der Lunge durch die Applikation von AAT-MSCs signifikant vermindert werden konnte, was auf eine Regeneration des Lungengewebes hindeutet. Auch in dieser Auswertung fiel der durch die Behandlung mit nicht-modifizierten MSCs erzielte Effekt geringer aus, was eine additive Wirkung von AAT und MSCs nahelegt. Zusammenfassend kann festgestellt werden, dass die vorliegende Arbeit einen ersten Nachweis des Wirkkonzepts von AAT-MSCs für die Behandlung von entzündlichen Lungenerkrankungen wie emphysematöser COPD erbringen konnte

Mesenchymal stem/stromal cells engineered to express the protease inhibitor alpha-1 antitrypsin for the treatment of inflammatory lung diseases

Chronic inflammatory diseases are a growing global health problem. Chronic obstructive pulmonary disease (COPD) represents a major cause of morbidity and mortality, and is currently the third leading cause of death worldwide. COPD is a devastating progressive lung disease characterized by airflow limitation, chronic bronchitis and bronchiolitis, emphysema, and small airway remodeling. There are no curative options for COPD to date, thus there is a desperate need for novel approaches and treatments. To this end, the goal of this thesis work was to develop and evaluate a novel cell product composed of mesenchymal stem/stromal cells (MSCs) genetically modified to express the protease inhibitor alpha-1 antitrypsin (AAT), thus combining the potent immunomodulatory and cytoprotective properties of MSCs, and of AAT. A major focus was the development of an optimized expression construct/vector system for MSC engineering. Eight versions of gamma-retroviral-based vectors were designed and tested for optimal transgenic expression of human AAT and puromycin-N-acetyltransferase (pac) for antibiotic selection. The constructs differed in the gene promoter used for driving AAT expression, and in the relative position of the expression cassettes within the vector backbone. The vectors were then compared with regards to viral titers, transduction efficacy of primary human MSCs, AAT expression in the supernatant of transduced cells, and cell yield after antibiotic selection. The best results for all parameters were obtained when AAT was expressed from the human eukaryotic translation elongation factor 1 alpha 1 short (EFS) promoter, and the pac gene was linked via an internal ribosome entry site (IRES) sequence. This lead candidate expression cassette was then transferred to a lentiviral backbone to assess if transduction and AAT expression from MSCs could be further increased by using this viral system. While values were higher with the gamma-retroviral vector at low multiplicities of infection (MOIs), transduction reached a plateau and no further increase was possible even when higher MOIs were used. Transduction with the lentiviral construct, however, followed a clear dose-response, reaching transduction efficacies >80% at the highest MOI tested. In vitro functionality of the protease inhibitor AAT expressed from MSCs was confirmed using a neutrophil elastase inhibition assay. A process for large scale transduction and expansion of gene-modified MSCs was subsequently developed. To evaluate the potential therapeutic benefit of AAT-MSCs in vivo, a mouse model of elastase-induced emphysema was used. Treatment of the mice with AAT-MSCs resulted in significant improvement of pulmonary function parameters, while only a slight functional amelioration was observed after treatment with non-modified MSCs. Histopathologic examination of AAT-MSC-treated mice revealed a significant decrease of airspace enlargement, indicating regeneration of pulmonary tissue. Again, the effect was smaller after treatment with non-modified MSCs, demonstrating a more than additive effect of combining AAT and MSCs. In conclusion, the present study provides the first in vivo proof of concept for the treatment of emphysematous COPD using engineered AAT-MSCs.Chronisch entzündliche Erkrankungen sind heutzutage ein globales, im Steigen begriffenes Gesundheitsproblem. Ein wichtiges Beispiel ist die chronisch obstruktive Lungenerkrankung (COPD), eine verheerende, fortschreitende Erkrankung der Lunge mit hoher Morbidität und Mortalität, gegenwärtig die dritthäufigste Todesursache weltweit. Symptome der COPD sind Atemnot, eine chronische Entzündung der Bronchien und Bronchiolen, die Entwicklung eines Emphysems und ein Umbau der engen Atemwege. Da bislang eine Heilung der COPD nicht möglich ist, besteht ein dringender Bedarf an innovativen Therapieansätzen. Wir entwickelten ein neuartiges Zellprodukt, basierend auf mesenchymalen Stamm-/Stromazellen (MSCs), die genetisch modifiziert wurden, sodass sie den Protease-Inhibitor Alpha-1 Antitrypsin (AAT) exprimieren. Diese Kombination ermöglicht es, die immunmodulatorischen und zytoprotektiven Eigenschaften der MSCs mit jenen von AAT in einem Produkt zu vereinen. Dabei wurde in der vorliegenden Arbeit ein Fokus auf die Entwicklung eines optimierten Expressionskonstruktes gelegt. Es wurden acht gamma-retrovirale Vektoren für die Expression von AAT und Puromycin-N-Acetyltransferase (pac), das eine Antibiotikaselektion genmodifizierter Zellen erlaubt, konzipiert und kloniert. Diese Konstrukte unterscheiden sich sowohl im Promoter, der für die AAT-Expression verwendet wird, als auch in der relativen Anordnung der Expressionskassetten im Plasmid. Die Auswahl des besten Konstrukts wurde anhand eines Vergleichs der Vektoren hinsichtlich der erzielten viralen Titer, der Transduktionseffizienz von primären humanen MSCs, des AAT-Gehalts im Zellkulturüberstand von transduzierten MSCs sowie der Zellausbeute nach Antibiotikaselektion durchgeführt. Die jeweils besten Ergebnisse wurden erzielt, wenn AAT von der kurzen Form des Promoters des humanen eukaryotischen Translationselongationsfaktors 1 alpha 1 (EFS) exprimiert wurde, und das pac Gen durch eine interne ribosomale Eintrittsstelle- (IRES)-Sequenz verknüpft war. Diese favorisierte Expressionskassette wurde in ein lentivirales Plasmid transferiert, um zu untersuchen, ob Transduktion und AAT-Expression in einem solchen System weiter verbessert werden können. Es zeigte sich, dass mittels des gamma-retroviralen Vektors größere Transduktionseffizienzen und AAT-Mengen bei niedriger Multiplizität der Infektion (MOI) erzielt werden konnten, aber bei höheren MOIs keine zusätzliche Verbesserung zu erreichen war. Im Gegensatz dazu folgten Transduktion und AAT-Expression mittels des lentiviralen Vektors einer eindeutigen Dosis-Wirkung, die bei hohen MOIs in Transduktionseffizienzen von >80% resultierte. Ein Neutrophile Elastase Inhibitionstest bestätigte die in vitro Funktionalität von MSC-sezerniertem AAT als Proteaseinhibitor, bevor Transduktions- und Expansionsprozesse zur Herstellung von AAT-MSCs im Großformat etabliert wurden. Um die therapeutische Wirkung von AAT-MSCs in vivo zu untersuchen, wurde ein Mausmodell eines Elastase-induzierten Emphysems durchgeführt. Es zeigte sich, dass die Behandlung von Mäusen mit AAT-MSCs zu einer signifikanten Verbesserung der Lungenfunktion der Tiere führte, während nicht-modifizierte MSCs nur eine geringfügige Wiederherstellung der pulmonalen Funktion bewirkten. Die histopathologische Analyse ergab, dass die Überblähung der Lunge durch die Applikation von AAT-MSCs signifikant vermindert werden konnte, was auf eine Regeneration des Lungengewebes hindeutet. Auch in dieser Auswertung fiel der durch die Behandlung mit nicht-modifizierten MSCs erzielte Effekt geringer aus, was eine additive Wirkung von AAT und MSCs nahelegt. Zusammenfassend kann festgestellt werden, dass die vorliegende Arbeit einen ersten Nachweis des Wirkkonzepts von AAT-MSCs für die Behandlung von entzündlichen Lungenerkrankungen wie emphysematöser COPD erbringen konnte

Targeting cardiac arrhythmia by enhancing mitochondrial calcium uptake

Cardiovascular diseases remain the number one cause for morbidity and mortality worldwide with an estimated half of cardiovascular disease-related deaths being attributed to cardiac arrhythmia. Despite this enormous importance for public health, existing antiarrhythmic drugs are still far from being ideal as they display perilous side effects and can not be administered over extended time periods. It is thus a major endeavor of cardiovascular research to identify novel safer drug targets and therapeutic strategies for the treatment of cardiac arrhythmia. Since cardiac rhythmicity is directly dependent on a tight regulation of intracellular Ca2+ and cardiac arrhythmia is often associated with disturbances in Ca2+ homeostasis, we used an unbiased approach to identify novel regulators of cardiac Ca2+ handling and modulators thereof. A library of newly synthesized, organic-like compounds was screened for their ability to restore rhythmic cardiac contractions in a zebrafish model for cardiac fibrillation. From this screen we identified the small ester compound efsevin, which binds to the voltage-dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane. We demonstrated that treatment with efsevin enhances mitochondrial Ca2+ uptake and thereby prevents propagation of spontaneous intracellular Ca2+ release events in cardiomyocytes, the triggers for ectopic excitations and arrhythmia. Since this work presented a novel role for VDAC2 in cardiomyocytes we analyzed the structure of VDAC2 by crystallography to identify elements that promote specificity of this isoform over VDAC1 and VDAC3. Though we could not detect large structural differences, we identified moieties that interact with regulatory proteins, which differ between the isoforms, and could thus explain the distinct role of VDAC2 in cardiomyocytes. We then used the crystal structure of VDAC2 to identify the binding site of efsevin by computational modeling and identified a binding pocket located between the wall of the VDAC2 pore and the pore-lining α helix, that was previously suggested to promote channel gating. In planar lipid bilayers we demonstrated that efsevin promotes gating of the channel from an anion-selective high conductance state into a more cation-selective low conductance state, thereby explaining the enhanced mitochondrial Ca2+ uptake induced by efsevin. To analyze the translational potential of efsevin, we tested efsevin in experimental models for the human cardiac arrhythmia catecholaminergic polymorphic ventricular tachycardia (CPVT). Efsevin reduced spontaneous diastolic Ca2+ signals and action potentials in cardiomyocytes isolated from CPVT mice and significantly reduced episodes of ventricular tachycardia in in vivo. Furthermore, efsevin reduced spontaneous, diastolic Ca2+ signals in induced pluripotent stem cell derived cardiomyocytes from a CPVT patient. Because efsevin lacks several features essential for druggability like e.g. a nanomolar affinity to the target and key pharmacokinetic properties like oral bioavailability, we then screened a library of clinically approved compounds for additional mitochondrial Ca2+ uptake enhancers. We identified increased uptake of Ca2+ into mitochondria of cardiomyocytes upon treatment with either the cholesterol uptake inhibitor ezetimbe or disulfiram, used for the treatment of alcohol abuse. Both were active at significantly lower concentrations compared to efsevin and showed efficacy experimental models for cardiac arrhythmia. Taken together, this thesis (i) establishes the outer mitochondrial membrane as a regulated barrier for Ca2+, (ii) establishes mitochondrial Ca2+ uptake as a novel regulator of cardiac rhythmicity and (iii) provides a novel candidate structure and lead substances for the development of a treatment for human cardiac arrhythmia

TRPM6 and TRPM7 differentially contribute to the relief of heteromeric TRPM6/7 channels from inhibition by cytosolic Mg2+ and Mg center dot ATP

TRPM6 and its homologue TRPM7 are alpha-kinase-coupled divalent cation-selective channels activated upon reduction of cytosolic levels of Mg2+ and Mg center dot ATP. TRPM6 is vital for organismal Mg2+ balance. However, mechanistically the cellular role and functional nonredundancy of TRPM6 remain incompletely understood. Comparative analysis of native currents in primary cells from TRPM6-versus TRPM7-deficient mice supported the concept that native TRPM6 primarily functions as a constituent of heteromeric TRPM6/7 channels. However, heterologous expression of the human TRPM6 protein engendered controversial results with respect to channel characteristics including its regulation by Mg2+ and Mg center dot ATP. To resolve this issue, we cloned the mouse TRPM6 (mTRPM6) cDNA and compared its functional characteristics to mouse TRPM7 (mTRPM7) after heterologous expression. Notably, we observed that mTRPM6 and mTRPM7 differentially regulate properties of heteromeric mTRPM6/7 channels: In the presence of mTRPM7, the extreme sensitivity of functionally expressed homomeric mTRPM6 to Mg2+ is tuned to higher concentrations, whereas mTRPM6 relieves mTRPM7 from the tight inhibition by Mg center dot ATP. Consequently, the association of mTRPM6 with mTRPM7 allows for high constitutive activity of mTRPM6/7 in the presence of physiological levels of Mg2+ and Mg center dot ATP, thus laying the mechanistic foundation for constant vectorial Mg2+ transport specifically into epithelial cells

The antiarrhythmic compound efsevin directly modulates voltage‐dependent anion channel 2 by binding to its inner wall and enhancing mitochondrial Ca2+ uptake

Background and Purpose The synthetic compound efsevin was recently identified to suppress arrhythmogenesis in models of cardiac arrhythmia, making it a promising candidate for antiarrhythmic therapy. Its activity was shown to be dependent on the voltage‐dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane. Here, we investigated the molecular mechanism of the efsevin–VDAC2 interaction. Experimental Approach To evaluate the functional interaction of efsevin and VDAC2, we measured currents through recombinant VDAC2 in planar lipid bilayers. Using molecular ligand‐protein docking and mutational analysis, we identified the efsevin binding site on VDAC2. Finally, physiological consequences of the efsevin‐induced modulation of VDAC2 were analysed in HL‐1 cardiomyocytes. Key Results In lipid bilayers, efsevin reduced VDAC2 conductance and shifted the channel's open probability towards less anion‐selective closed states. Efsevin binds to a binding pocket formed by the inner channel wall and the pore‐lining N‐terminal α‐helix. Exchange of amino acids N207, K236 and N238 within this pocket for alanines abolished the channel's efsevin‐responsiveness. Upon heterologous expression in HL‐1 cardiomyocytes, both channels, wild‐type VDAC2 and the efsevin‐insensitive VDAC2AAA restored mitochondrial Ca2+ uptake, but only wild‐type VDAC2 was sensitive to efsevin. Conclusion and Implications In summary, our data indicate a direct interaction of efsevin with VDAC2 inside the channel pore that leads to modified gating and results in enhanced SR‐mitochondria Ca2+ transfer. This study sheds new light on the function of VDAC2 and provides a basis for structure‐aided chemical optimization of efsevin

Mitochondrial Ca(2+) uptake by the voltage-dependent anion channel 2 regulates cardiac rhythmicity.

Tightly regulated Ca(2+) homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca(2+) handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca(2+) extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca(2+) uptake and accelerates the transfer of Ca(2+) from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca(2+) sparks and thereby inhibits Ca(2+) overload-induced erratic Ca(2+) waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin\u27s rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca(2+) uptake in the regulation of cardiac rhythmicity

Ten Years of High Resolution Structural Research on the Voltage Dependent Anion Channel (VDAC)—Recent Developments and Future Directions

Mitochondria are evolutionarily related to Gram-negative bacteria and both comprise two membrane systems with strongly differing protein composition. The major protein in the outer membrane of mitochondria is the voltage-dependent anion channel (VDAC), which mediates signal transmission across the outer membrane but also the exchange of metabolites, most importantly ADP and ATP. More than 30 years after its discovery three identical high-resolution structures were determined in 2008. These structures show a 19-stranded anti-parallel beta-barrel with an N-terminal helix located inside. An odd number of beta-strands is also shared by Tom40, another member of the VDAC superfamily. This indicates that this superfamily is evolutionarily relatively young and that it has emerged in the context of mitochondrial evolution. New structural information obtained during the last decade on Tom40 can be used to cross-validate the structure of VDAC and vice versa. Interpretation of biochemical and biophysical studies on both protein channels now rests on a solid basis of structural data. Over the past 10 years, complementary structural and functional information on proteins of the VDAC superfamily has been collected from in-organello, in-vitro, and in silico studies. Most of these findings have confirmed the validity of the original structures. This short article briefly reviews the most important advances on the structure and function of VDAC superfamily members collected during the last decade and summarizes how they enhanced our understanding of the channel

Effects of inserting fluorescent proteins into the α1S II–III loop: insights into excitation–contraction coupling

In skeletal muscle, intermolecular communication between the 1,4-dihydropyridine receptor (DHPR) and RYR1 is bidirectional: orthograde coupling (skeletal excitation–contraction coupling) is observed as depolarization-induced Ca2+ release via RYR1, and retrograde coupling is manifested by increased L-type Ca2+ current via DHPR. A critical domain (residues 720–765) of the DHPR α1S II–III loop plays an important but poorly understood role in bidirectional coupling with RYR1. In this study, we examine the consequences of fluorescent protein insertion into different positions within the α1S II–III loop. In four constructs, a cyan fluorescent protein (CFP)–yellow fluorescent protein (YFP) tandem was introduced in place of residues 672–685 (the peptide A region). All four constructs supported efficient bidirectional coupling as determined by the measurement of L-type current and myoplasmic Ca2+ transients. In contrast, insertion of a CFP–YFP tandem within the N-terminal portion of the critical domain (between residues 726 and 727) abolished bidirectional signaling. Bidirectional coupling was partially preserved when only a single YFP was inserted between residues 726 and 727. However, insertion of YFP near the C-terminal boundary of the critical domain (between residues 760 and 761) or in the conserved C-terminal portion of the α1S II–III loop (between residues 785 and 786) eliminated bidirectional coupling. None of the fluorescent protein insertions, even those that interfered with signaling, significantly altered membrane expression or targeting. Thus, bidirectional signaling is ablated by insertions at two different sites in the C-terminal portion of the α1S II–III loop. Significantly, our results indicate that the conserved portion of the α1S II–III loop C terminal to the critical domain plays an important role in bidirectional coupling either by conveying conformational changes to the critical domain from other regions of the DHPR or by serving as a site of interaction with other junctional proteins such as RYR1

Identification and expression of voltage-gated calcium channel Β subunits in Zebrafish

Voltage-gated calcium channels (VGCC) play important roles in electrically excitable cells and embryonic development. The VGCC Β subunits are essential for membrane localization of the channel and exert modulatory effects on channel functions. In mammals, the VGCC Β subunit gene family contains four members. In zebrafish, there appear to be seven VGCC Β subunits including the previously identified Β1 subunit. cDNAs for six additional VGCC Β subunit homologs were identified in zebrafish, their chromosomal locations determined and their expression patterns characterized during embryonic development. These six genes are primarily expressed in the nervous system with cacnb4a also expressed in the developing heart. Sequence homology, genomic synteny and expression patterns suggest that there are three pairs of duplicate genes for Β2, Β3, and Β4 in zebrafish with distinct expression patterns during embryonic development. Developmental Dynamics 237:3842–3852, 2008. © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61457/1/21776_ftp.pd