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Geoffrey W Abbott - One of the best experts on this subject based on the ideXlab platform.
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KCNQ and KCNE Isoform-Dependent Pharmacology Rationalizes Native American Dual Use of Specific Plants as Both Analgesics and Gastrointestinal Therapeutics
'Frontiers Media SA', 2021Co-Authors: Geoffrey W Abbott, Kaitlyn E. Redford, Ryan F. Yoshimura, Rían W. Manville, Luiz Moreira, Kevin Tran, Grey Arena, Alexandra Kookootsedes, Emma Lasky, Elliot GunnisonAbstract:Indigenous peoples of the Americas are proficient in botanical medicine. KCNQ family voltage-gated potassium (Kv) channels are sensitive to a variety of ligands, including plant metabolites. Here, we screened methanolic extracts prepared from 40 Californian coastal redwood forest plants for effects on Kv current and membrane potential in Xenopus oocytes heterologously expressing KCNQ2/3, which regulates excitability of neurons, including those that sense pain. Extracts from 9 of the 40 plant species increased KCNQ2/3 current at –60 mV by ≥threefold (maximally, 15-fold by Urtica dioica) and/or hyperpolarized membrane potential by ≥-3 mV (maximally, –11 mV by Arctostaphylos glandulosa). All nine plants have traditionally been used as both analgesics and gastrointestinal therapeutics. Of two extracts tested, both acted as KCNQ-dependent analgesics in mice. KCNQ2/3 activation at physiologically relevant, subthreshold membrane potentials by tannic acid, gallic acid and quercetin provided molecular correlates for analgesic action of several of the plants. While tannic acid also activated KCNQ1 and KCNQ1-KCNE1 at hyperpolarized, negative membrane potentials, it inhibited KCNQ1-KCNE3 at both negative and positive membrane potentials, mechanistically rationalizing historical use of tannic acid-containing plants as gastrointestinal therapeutics. KCNE dependence of KCNQ channel modulation by plant metabolites therefore provides a molecular mechanistic basis for Native American use of specific plants as both analgesics and gastrointestinal aids
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the kcne2 potassium channel β subunit is required for normal lung function and resilience to ischemia and reperfusion injury
The FASEB Journal, 2019Co-Authors: Leng Zhou, Torsten K Roepke, Clemens Kohncke, Geoffrey W AbbottAbstract:The KCNE2 single transmembrane-spanning voltage-gated potassium (Kv) channel β subunit is ubiquitously expressed and essential for normal function of a variety of cell types, often via regulation of the KCNQ1 Kv channel. A polymorphism upstream of KCNE2 is associated with reduced lung function in human populations, but the pulmonary consequences of KCNE2 gene disruption are unknown. Here, germline deletion of mouse Kcne2 reduced pulmonary expression of potassium channel α subunits Kcnq1 and Kcnb1 but did not alter expression of other Kcne genes. Kcne2 colocalized and coimmunoprecipitated with Kcnq1 in mouse lungs, suggesting the formation of pulmonary Kcnq1-Kcne2 potassium channel complexes. Kcne2 deletion reduced blood O2, increased CO2, increased pulmonary apoptosis, and increased inflammatory mediators TNF-α, IL-6, and leukocytes in bronchoalveolar lavage (BAL) fluids. Consistent with increased pulmonary vascular leakage, Kcne2 deletion increased plasma, BAL albumin, and the BAL:plasma albumin concentration ratio. Kcne2-/- mouse lungs exhibited baseline induction of the reperfusion injury salvage kinase pathway but were less able to respond via this pathway to imposed pulmonary ischemia/reperfusion injury (IRI). We conclude that KCNE2 regulates KCNQ1 in the lungs and is required for normal lung function and resistance to pulmonary IRI. Our data support a causal relationship between KCNE2 gene disruption and lung dysfunction.-Zhou, L., Kohncke, C., Hu, Z., Roepke, T. K., Abbott, G. W. The KCNE2 potassium channel β subunit is required for normal lung function and resilience to ischemia and reperfusion injury.
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β Subunits Control the Effects of Human Kv4.3 Potassium Channel Phosphorylation
Frontiers Media S.A., 2017Co-Authors: Geoffrey W AbbottAbstract:The transient outward K+ current, Ito, activates early in the cardiac myocyte action potential, to begin repolarization. Human Ito is generated primarily by two Kv4.3 potassium channel α subunit splice variants (Kv4.3L and Kv4.3S) that diverge only by a C-terminal, membrane-proximal, 19-residue stretch unique to Kv4.3L. Protein kinase C (PKC) phosphorylation of threonine 504 within the Kv4.3L-specific 19-residues mediates α-adrenergic inhibition of Ito in human heart. Kv4.3 is regulated in human heart by various β subunits, including cytosolic KChIP2b and transmembrane KCNEs, yet their impact on the functional effects of human Kv4.3 phosphorylation has not been reported. Here, this gap in knowledge was addressed using human Kv4.3 splice variants, T504 mutants, and human β subunits. Subunits were co-expressed in Xenopus laevis oocytes and analyzed by two-electrode voltage-clamp, using phorbol 12-myristate 13-acetate (PMA) to stimulate PKC. Unexpectedly, KChIP2b removed the inhibitory effect of PKC on Kv4.3L (but not Kv4.3L threonine phosphorylation by PKC per-se), while co-expression with KCNE2, but not KCNE4, restored PKC-dependent inhibition of Kv4.3L-KChIP2b to quantitatively resemble previously reported effects of α-adrenergic modulation of human ventricular Ito. In addition, PKC accelerated recovery from inactivation of Kv4.3L-KChIP2b channels and, interestingly, of both Kv4.3L and Kv4.3S alone. Thus, β subunits regulate the response of human Kv4.3 to PKC phosphorylation and provide a potential mechanism for modifying the response of Ito to α-adrenergic regulation in vivo
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kcne1 and KCNE3 the yin and yang of voltage gated k channel regulation
Gene, 2016Co-Authors: Geoffrey W AbbottAbstract:Abstract The human KCNE gene family comprises five genes encoding single transmembrane-spanning ion channel regulatory subunits. The primary function of KCNE subunits appears to be regulation of voltage-gated potassium (Kv) channels, and the best-understood KCNE complexes are with the KCNQ1 Kv α subunit. Here, we review the often opposite effects of KCNE1 and KCNE3 on Kv channel biology, with an emphasis on regulation of KCNQ1. Slow-activating I Ks channel complexes formed by KCNQ1 and KCNE1 are essential for human ventricular myocyte repolarization, while constitutively active KCNQ1-KCNE3 channels are important in the intestine. Inherited sequence variants in human KCNE1 and KCNE3 cause cardiac arrhythmias but by different mechanisms, and each is important for hearing in unique ways. Because of their contrasting effects on KCNQ1 function, KCNE1 and KCNE3 have proved invaluable tools in the mechanistic understanding of how channel gating can be manipulated, and each may also provide a window into novel insights and new therapeutic opportunities in K + channel pharmacology. Finally, findings from studies of Kcne1 −/− and KCNE3 −/− mouse lines serve to illustrate the complexity of KCNE biology and KCNE-linked disease states.
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kcne1 and KCNE3 the yin and yang of voltage gated k channel regulation
Gene, 2016Co-Authors: Geoffrey W AbbottAbstract:Abstract The human KCNE gene family comprises five genes encoding single transmembrane-spanning ion channel regulatory subunits. The primary function of KCNE subunits appears to be regulation of voltage-gated potassium (Kv) channels, and the best-understood KCNE complexes are with the KCNQ1 Kv α subunit. Here, we review the often opposite effects of KCNE1 and KCNE3 on Kv channel biology, with an emphasis on regulation of KCNQ1. Slow-activating I Ks channel complexes formed by KCNQ1 and KCNE1 are essential for human ventricular myocyte repolarization, while constitutively active KCNQ1-KCNE3 channels are important in the intestine. Inherited sequence variants in human KCNE1 and KCNE3 cause cardiac arrhythmias but by different mechanisms, and each is important for hearing in unique ways. Because of their contrasting effects on KCNQ1 function, KCNE1 and KCNE3 have proved invaluable tools in the mechanistic understanding of how channel gating can be manipulated, and each may also provide a window into novel insights and new therapeutic opportunities in K + channel pharmacology. Finally, findings from studies of Kcne1 −/− and KCNE3 −/− mouse lines serve to illustrate the complexity of KCNE biology and KCNE-linked disease states.
Charles R Sanders - One of the best experts on this subject based on the ideXlab platform.
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Structural Investigation of the Transmembrane Domain of KCNE1 in Proteoliposomes
2015Co-Authors: Indra D Sahu, Megan M. Dunagan, Brett M Kroncke, Rongfu Zhang, Robert M Mccarrick, Hubbell J Smith, Charles R Sanders, Andrew Craig, Gary A LoriganAbstract:KCNE1 is a single-transmembrane protein of the KCNE family that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden infant death syndrome, and deafness. The transmembrane domain (TMD) of KCNE1 plays a key role in mediating the physical association with KCNQ1 and in subsequent modulation of channel gating kinetics and conductance. However, the mechanisms associated with these roles for the TMD remain poorly understood, highlighting a need for experimental structural studies. A previous solution NMR study of KCNE1 in LMPG micelles revealed a curved transmembrane domain, a structural feature proposed to be critical to KCNE1 function. However, this curvature potentially reflects an artifact of working in detergent micelles. Double electron electron resonance (DEER) measurements were conducted on KCNE1 in LMPG micelles, POPC/POPG proteoliposomes, and POPC/POPG lipodisq nanoparticles to directly compare the structure of the TMD in a variety of different membrane environments. Experimentally derived DEER distances coupled with simulated annealing molecular dynamic simulations were used to probe the bilayer structure of the TMD of KCNE1. The results indicate that the structure is helical in proteoliposomes and is slightly curved, which is consistent with the previously determined solution NMR structure in micelles. The evident resilience of the curvature in the KCNE1 TMD leads us to hypothesize that the curvature is likely to be maintained upon binding of the protein to the KCNQ1 channel
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structural investigation of the transmembrane domain of kcne1 in proteoliposomes
Biochemistry, 2014Co-Authors: Indra D Sahu, Megan M. Dunagan, Brett M Kroncke, Andrew F Craig, Rongfu Zhang, Robert M Mccarrick, Hubbell J Smith, Charles R Sanders, Gary A LoriganAbstract:KCNE1 is a single-transmembrane protein of the KCNE family that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden infant death syndrome, and deafness. The transmembrane domain (TMD) of KCNE1 plays a key role in mediating the physical association with KCNQ1 and in subsequent modulation of channel gating kinetics and conductance. However, the mechanisms associated with these roles for the TMD remain poorly understood, highlighting a need for experimental structural studies. A previous solution NMR study of KCNE1 in LMPG micelles revealed a curved transmembrane domain, a structural feature proposed to be critical to KCNE1 function. However, this curvature potentially reflects an artifact of working in detergent micelles. Double electron electron resonance (DEER) measurements were conducted on KCNE1 in LMPG micelles, POPC/POPG proteoliposomes, and POPC/POPG lipodis...
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structural investigation of the transmembrane domain of kcne1 in proteoliposomes
Biochemistry, 2014Co-Authors: Indra D Sahu, Megan M. Dunagan, Brett M Kroncke, Andrew F Craig, Rongfu Zhang, Robert M Mccarrick, Hubbell J Smith, Charles R Sanders, Gary A LoriganAbstract:KCNE1 is a single-transmembrane protein of the KCNE family that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden infant death syndrome, and deafness. The transmembrane domain (TMD) of KCNE1 plays a key role in mediating the physical association with KCNQ1 and in subsequent modulation of channel gating kinetics and conductance. However, the mechanisms associated with these roles for the TMD remain poorly understood, highlighting a need for experimental structural studies. A previous solution NMR study of KCNE1 in LMPG micelles revealed a curved transmembrane domain, a structural feature proposed to be critical to KCNE1 function. However, this curvature potentially reflects an artifact of working in detergent micelles. Double electron electron resonance (DEER) measurements were conducted on KCNE1 in LMPG micelles, POPC/POPG proteoliposomes, and POPC/POPG lipodis...
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a model of human potassium channel kcnq1 modulation by accessory protein KCNE3
Biophysical Journal, 2014Co-Authors: Brett M Kroncke, D P Nannemann, Jens Meiler, Wade D Van Horn, Carlos G. Vanoye, Charles R SandersAbstract:Human potassium channel KCNQ1 is expressed in several tissues including inner ear, heart muscle, lung, intestine, and stomach, each requiring a unique current profile for proper function. To tune its current output, KCNQ1 complexes with several accessory proteins from the KCNE family. Each KCNE family member modulates KCNQ1 differently: KCNE1 causes the channel to delay opening and become more conductive in the open state, KCNE3 causes the channel to be constitutively open and more conductive, and KCNE4 causes the channel to close. To extend previous efforts to characterize the spectrum of KCNQ1 structure and modulation, we used a hybrid experimental-computational approach to model the KCNQ1-KCNE3 complex. Our strategy was to determine the NMR structure of KCNE3 alone in lipid-mimicking bicelles, build a homology model of KCNQ1, generate in vivo restraints using electrophysiology, and dock the structure of KCNE3 onto the homology model of KCNQ1 using electrophysiology-based restraints to validate and refine the resulting models.The results from this method will be presented.
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kcnq1 kcne1 assembly co translation not required
Channels, 2010Co-Authors: Carlos G. Vanoye, Richard C Welch, Charles R Sanders, Changlin Tian, Alfred L GeorgeAbstract:Voltage-gated potassium channels are often assembled with accessory proteins which increases their functional diversity. KCNE proteins are small accessory proteins that modulate voltage-gated potassium (KV) channels. Although the functional effects of various KCNE proteins have been described, many questions remain regarding their assembly with the pore-forming subunits. For example, while previous experiments with some KV channels suggest that the association of the pore-subunit with the accessory subunits occurs co-translationally in the endoplasmic reticulum, it is not known whether KCNQ1 assembly with KCNE1 occurs in a similar manner to generate the medically important cardiac slow delayed rectifier current (IKs). In this study we used a novel approach to demonstrate that purified recombinant human KCNE1 protein (prKCNE1) modulates KCNQ1 channels heterologously expressed in Xenopus oocytes resulting in generation of IKs. Incubation of KCNQ1-expressing oocytes with cycloheximide did not prevent IKs exp...
Antonio Felipe - One of the best experts on this subject based on the ideXlab platform.
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Calmodulin-dependent KCNE4 dimerization controls membrane targeting
'Springer Science and Business Media LLC', 2021Co-Authors: Sara R. Roig, Laura Solé, Silvia Cassinelli, Magalí Colomer-molera, Daniel Sastre, Clara Serrano-novillo, Antonio Serrano-albarrás, Pilar M. Lillo, Michael M. Tamkun, Antonio FelipeAbstract:Abstract The voltage-dependent potassium channel Kv1.3 participates in the immune response. Kv1.3 is essential in different cellular functions, such as proliferation, activation and apoptosis. Because aberrant expression of Kv1.3 is linked to autoimmune diseases, fine-tuning its function is crucial for leukocyte physiology. Regulatory KCNE subunits are expressed in the immune system, and KCNE4 specifically tightly regulates Kv1.3. KCNE4 modulates Kv1.3 currents slowing activation, accelerating inactivation and retaining the channel at the endoplasmic reticulum (ER), thereby altering its membrane localization. In addition, KCNE4 genomic variants are associated with immune pathologies. Therefore, an in-depth knowledge of KCNE4 function is extremely relevant for understanding immune system physiology. We demonstrate that KCNE4 dimerizes, which is unique among KCNE regulatory peptide family members. Furthermore, the juxtamembrane tetraleucine carboxyl-terminal domain of KCNE4 is a structural platform in which Kv1.3, Ca2+/calmodulin (CaM) and dimerizing KCNE4 compete for multiple interaction partners. CaM-dependent KCNE4 dimerization controls KCNE4 membrane targeting and modulates its interaction with Kv1.3. KCNE4, which is highly retained at the ER, contains an important ER retention motif near the tetraleucine motif. Upon escaping the ER in a CaM-dependent pattern, KCNE4 follows a COP-II-dependent forward trafficking mechanism. Therefore, CaM, an essential signaling molecule that controls the dimerization and membrane targeting of KCNE4, modulates the KCNE4-dependent regulation of Kv1.3, which in turn fine-tunes leukocyte physiology
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Selective Formation of Oligomeric Kv7.5 (KCNQ5)/KCNE1 and Kv7.5 (KCNQ5)/KCNE3 Channels. Differential Targeting to Membrane Surface Microdomains
Biophysical Journal, 2012Co-Authors: Laura Solé, Alvaro Villarroel, Sara R. Roig, Meritxell Roura-ferrer, Anna Oliveras, Núria Comes, Albert Vallejo, Antonio FelipeAbstract:Kv7 (KCNQ) proteins form a family of voltage-gated potassium channels that is comprised of five members, Kv7.1-Kv7.5. While Kv7.1 is crucial in the heart, the Kv7.2-Kv7.5 channels contribute to the M-current in the nervous system. In addition, Kv7.5 is expressed in muscles, where its physiological role is currently under evaluation. Kv7 associations with KCNE accessory subunits (KCNE1-5) enhance channel diversity. KCNE peptides control the surface expression, voltage-dependence, kinetics of gating, unitary conductance, ion selectivity and pharmacology of several channels. KCNE subunits have been primarily studied in the heart; however, their activity in the brain and in many other tissues is being increasingly recognized. Here, we found that Kv7.5 and KCNE subunits are present in myoblasts. Therefore, oligomeric associations may underlie some Kv7.5 functional diversity in skeletal muscle. Expression in Xenopus oocytes and HEK-293 cells demonstrates that KCNE1 and KCNE3, but none of the other KCNE subunits, associate to Kv7.5. While KCNE1 slows activation and suppresses inward rectification, KCNE3 inhibits Kv7.5 currents. Furthermore, KCNE1 increases Kv7.5 currents in HEK cells. The membrane targeting is also affected. Biochemical isolation of lipid rafts demonstrates that Kv7.5 barely locates in rafts. KCNE1 and KCNE3 show differential targeting. While KCNE3 targets to rafts, KCNE1 does not. Association of Kv7.5 with KCNE3 impairs KCNE3 targeting to rafts and this is further supported by FRAP analysis. Our results have physiological relevance since Kv7.5 is abundant in skeletal and smooth muscle and its association with KCNE peptides may fine-tune cellular responses.Supported by the Ministerio de Ciencia e Innovacion (MICINN), Spain (BFU2005-00695, BFU2008-00431 and CSD2008-00005).
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Targeting of Kv7.5 (KCNQ5)/KCNE channels to surface microdomains of cell membranes.
Muscle & nerve, 2011Co-Authors: Meritxell Roura-ferrer, Alvaro Villarroel, Laura Solé, Anna Oliveras, Núria Comes, Antonio FelipeAbstract:Background: Kv7.5 (KCNQ5) channels conduct M-type potassium currents in the brain, are expressed in skeletal muscle, and contribute to vascular muscle tone. Methods: We coexpressed Kv7.5 and KCNE1–3 peptides in HEK293 cells and then analyzed their association using electrophysiology and co-immunoprecipitation, assessed localization using confocal microscopy, examined targeting of the oligomeric channels to cholesterol-rich membrane surface microdomains using lipid raft isolation, and evaluated their membrane dynamics using fluorescence recovery after photobleaching (FRAP). Results: Kv7.5 forms oligomeric channels specifically with KCNE1 and KCNE3. The expression of Kv7.5 targeted to cholesterol-rich membrane surface microdomains was very low. Oligomeric Kv7.5/KCNE1 and Kv7.5/KCNE3 channels did not localize to lipid rafts. However, Kv7.5 association impaired KCNE3 expression in lipid raft microdomains. Conclusions: Our results indicate that Kv7.5 contributes to the spatial regulation of KCNE3. This new scenario could greatly assist in determining the physiological relevance of putative KCNE3 interactions in nerve and muscle. Muscle Nerve 45: 48–54, 2012
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Functional implications of KCNE subunit expression for the Kv7.5 (KCNQ5) channel.
Cellular physiology and biochemistry : international journal of experimental cellular physiology biochemistry and pharmacology, 2009Co-Authors: Meritxell Roura-ferrer, Alvaro Villarroel, Laura Solé, Anna Oliveras, Núria Comes, Ainhoa Etxebarria, Antonio FelipeAbstract:Kv7 (KCNQ) proteins form a family of voltage-gated potassium channels that is comprised of five members, Kv7.1-Kv7.5. While Kv7.1 is crucial in the heart, the Kv7.2, Kv7.3, Kv7.4 and Kv7.5 channels contribute to the M-current in the nervous system. In addition to the brain, Kv7.5 is expressed in skeletal and smooth muscle, where its physiological role is currently under evaluation. Kv7 associations with KCNE accessory subunits (KCNE1-5) enhance channel diversity and their interaction provides mechanisms to respond to a variety of stimuli. KCNE peptides control the surface expression, voltage-dependence, kinetics of gating, unitary conductance, ion selectivity and pharmacology of several channels. KCNE subunits have been primarily studied in the heart; however, their activity in the brain and in many other tissues is being increasingly recognized. Here, we found that Kv7.5 and KCNE subunits are present in myoblasts. Therefore, oligomeric associations may underlie some Kv7.5 functional diversity in skeletal muscle. An extensive study in Xenopus oocytes and HEK-293 cells demonstrates that KCNE1 and KCNE3, but none of the other KCNE subunits, affect Kv7.5 currents. While KCNE1 slows activation and suppresses inward rectification, KCNE3 drastically inhibits Kv7.5 currents. In addition, KCNE1 increases Kv7.5 currents in HEK cells. Changes in gating and amplitude indicate functional interactions. Our results have physiological relevance since Kv7.5 is abundant in skeletal and smooth muscle and its association with KCNE peptides may fine-tune cellular responses.
Solé Codina I Laura - One of the best experts on this subject based on the ideXlab platform.
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Does a physiological role for KCNE subunits exist in the immune system?
'Informa UK Limited', 2020Co-Authors: Solé Codina I Laura, Felipe Campo AntonioAbstract:The study of channel modulation by regulatory subunits has attracted considerable attention. Evidence indicates a pivotal role for accessory proteins in the channelosome. For instance, these regulatory subunits are necessary to recapitulate in vivo ion currents and to further understand the physiological role of ion channels. KCNEs are a family of regulatory subunits that interact with a wide range of channels. We have described for the first time a molecular interaction between KCNE4 and the voltage-dependent potassium channel Kv1.3. The association of KCNE4, which alters the biophysical properties, trafficking and membrane localization of Kv1.3, functions as an endogenous dominant-negative mechanism. Since both proteins are expressed in the immune system, Kv1.3/KCNE4 channels may contribute to the fine-tuning of the immune response. Therefore, our results point to KCNE4 as a novel target for immunomodulation. KCNE4 is not the only KCNE which is expressed in leukocytes. All KCNEs (KCNE1-5) are present, and some members demonstrate modulation during proliferation and cancer. In summary, regulatory KCNE subunits are expressed in the immune system. In addition, several voltage-dependent K+ channels, which could interact with KCNEs, are also detected. Therefore, KCNE subunits may play a yet undiscovered role in the physiology of the immune system
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Role of KCN E4 on the voltage gated potassium channel Kv1.3 = Paper de KCNE4 en el canal de potassi dependent de voltage Kv1.3
'Edicions de la Universitat de Barcelona', 2014Co-Authors: Solé Codina I LauraAbstract:[cat] Els canals de potassi dependents de voltatge (Kv) juguen un paper molt important tant en cèl•lules excitables com no excitables. La possibilitat de formar hetero-oligomers i la d’associació amb subunitats reguladores són uns dels mecanismes que existeixen per tal de proveir de diferents mecanismes per a respondre de manera diferent enfront a canvis en el potencial de membrana. La composició del canalosoma modula tant la seva expressió a superfície com l’activitat d’aquests. Aquesta tesi es centra en l’estudi de l’efecte del la família de les subunitats reguladores KCNE sobre diferents Kv. Primerament s’estudià l’efecte que causaven en el tràfic del canal Kv7.1 (canal model per a l’estudi dels KCNEs) i posteriorment s’amplià l’estudi a un altre membre de la mateixa família, Kv7.5. A continuació s’estudià un canal d’elevada importància per a l’activació i proliferació leucocitària: Kv1.3, centrant-nos sobretot en l’efecte causat per un dels KCNEs: KCNE4. Aquesta subunitat no només inhibeix dràsticament el corrent del canal Kv1.3, sinó que a més a més, modifica el seu tràfic i localització. Aquests canvis són deguts a una interacció directa entre ambdues proteïnes. A continuació s’estudià en detall el complex Kv1.3-KCNE4. Mitjançant la combinació d’experiments d’electrofisiologia i monitorització de fluorescència de molècules individuals en la membrana, es va poder establir l’estequiometria d’aquest complex. Posteriorment, mitjançant l’anàlisi de diverses proteïnes quimèriques i mutants, tant del canal com de la subunitat reguladora, es van cercar els determinants moleculars implicats en l’associació entre ambdues proteïnes. S’han pogut determinar els motius claus en KCNE4 i Kv1.3 implicats en la formació del complex, però no en la modulació del canal. Finalment, degut a la importància de Kv1.3 en el sistema immunitari, s’han analitzat els nivells d’expressió dels KCNEs en diferents línies leucocitàries. S’ha observat que aquestes subunitats pateixen una regulació diferencial en funció de la manera d’activació i al llarg de la proliferació del leucòcits, suggerint un possible paper en la regulació precisa de la resposta immunològica.[eng] Voltage gated potassium channels (Kv) play important roles in different biological process such as generation and propagation of the nerve pulse and the cardiac action potential, promotion of insulin secretion, cell volume control, induction of cell proliferation, apoptosis, migration and initiation of many signaling pathways. Kv channels can homo- or hetero- tetramerize. The composition of the channel modulates their surface expression and serves as a mechanism for regulating channel activity. Kv channel interaction with accessory subunits provides mechanisms for channels to respond to stimuli beyond changes in membrane potential. The present dissertation is focused in the analysis of the effect of one regulatory subunits family (KCNEs) on different Kv channels. The first channel analyzed is Kv7.1, which is one of the most well-known channels to interact with all the KCNE family members. In fact KCNE1-Kv7.1 complex is focus of a huge number of studies, due to its important role in heart. Most of the studies though are focused to their electrophysiological properties and molecular determinants involved in the interaction. We performed traffic analysis experiments of Kv7.1 in the present of KCNE1-5 and demonstrated that Kv7.1 membrane surface localization is modified by some of them. Next, analysis was expanded to another channel from the same family, less characterized: Kv7.5. We demonstrated that from the five KCNEs members, only KCNE1 and KCNE3 modulate Kv7.5 activity. Furthermore, we demonstrated that Kv7.5 association to KCNE3 modifies the targeting of the regulatory subunit. Next, we moved to a non-related Kv channel such as Kv1.3, which plays a crucial role in the immune system. We first focused into characterizing the modulation of Kv1.3. We demonstrated that KCNE4, but not KCNE2, functions as an inhibitory Kv1.3 partner. Kv1.3 trafficking, targeting and activity are altered by the presence of KCNE4. Furthermore, by the combination of a plethora of approaches such as electrophysiological experiments from chimeric proteins and GFP single bleaching counting steps methodology we deciphered the stoichiometry of the Kv1.3-KCNE4 complex. Next, by immunoprecipitation experiments, traffic analysis and electrophysiological experiments, we analyzed the molecular determinants involved in the association between Kv1.3 and KCNE4. We have map a domain of Kv1.3 and a specific motif of KCNE4 involved in the formation of Kv1.3-KCNE4 complex, but not in the modulation of the channel. We also proposed a 3D docking model of Kv1.3 and KCNE4. Finally, due to the importance of Kv1.3 in the immune system, the expression of all the KCNE family has been analyzed in several cell lines of leukocytes. We have demonstrated that all KCNEs suffer a differential regulation among proliferation of leukocytes. Furthermore, a different regulation can be observed, depend on the mode of leukocytes’ activation. Our results further suggest a new and yet unidentified physiological role for KCNE subunits in the immune system. Putative associations of these ancillary proteins with Kv channels would yield a wide variety of biophysically and pharmacologically distinct channels that fine-tune the immunological response
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Role of KCN E4 on the voltage gated potassium channel Kv1.3 = Paper de KCNE4 en el canal de potassi dependent de voltage Kv1.3
'Edicions de la Universitat de Barcelona', 2013Co-Authors: Solé Codina I LauraAbstract:Voltage gated potassium channels (Kv) play important roles in different biological process such as generation and propagation of the nerve pulse and the cardiac action potential, promotion of insulin secretion, cell volume control, induction of cell proliferation, apoptosis, migration and initiation of many signaling pathways. Kv channels can homo- or hetero- tetramerize. The composition of the channel modulates their surface expression and serves as a mechanism for regulating channel activity. Kv channel interaction with accessory subunits provides mechanisms for channels to respond to stimuli beyond changes in membrane potential. The present dissertation is focused in the analysis of the effect of one regulatory subunits family (KCNEs) on different Kv channels. The first channel analyzed is Kv7.1, which is one of the most well-known channels to interact with all the KCNE family members. In fact KCNE1-Kv7.1 complex is focus of a huge number of studies, due to its important role in heart. Most of the studies though are focused to their electrophysiological properties and molecular determinants involved in the interaction. We performed traffic analysis experiments of Kv7.1 in the present of KCNE1-5 and demonstrated that Kv7.1 membrane surface localization is modified by some of them. Next, analysis was expanded to another channel from the same family, less characterized: Kv7.5. We demonstrated that from the five KCNEs members, only KCNE1 and KCNE3 modulate Kv7.5 activity. Furthermore, we demonstrated that Kv7.5 association to KCNE3 modifies the targeting of the regulatory subunit. Next, we moved to a non-related Kv channel such as Kv1.3, which plays a crucial role in the immune system. We first focused into characterizing the modulation of Kv1.3. We demonstrated that KCNE4, but not KCNE2, functions as an inhibitory Kv1.3 partner. Kv1.3 trafficking, targeting and activity are altered by the presence of KCNE4. Furthermore, by the combination of a plethora of approaches such as electrophysiological experiments from chimeric proteins and GFP single bleaching counting steps methodology we deciphered the stoichiometry of the Kv1.3-KCNE4 complex. Next, by immunoprecipitation experiments, traffic analysis and electrophysiological experiments, we analyzed the molecular determinants involved in the association between Kv1.3 and KCNE4. We have map a domain of Kv1.3 and a specific motif of KCNE4 involved in the formation of Kv1.3-KCNE4 complex, but not in the modulation of the channel. We also proposed a 3D docking model of Kv1.3 and KCNE4. Finally, due to the importance of Kv1.3 in the immune system, the expression of all the KCNE family has been analyzed in several cell lines of leukocytes. We have demonstrated that all KCNEs suffer a differential regulation among proliferation of leukocytes. Furthermore, a different regulation can be observed, depend on the mode of leukocytes’ activation. Our results further suggest a new and yet unidentified physiological role for KCNE subunits in the immune system. Putative associations of these ancillary proteins with Kv channels would yield a wide variety of biophysically and pharmacologically distinct channels that fine-tune the immunological response.Els canals de potassi dependents de voltatge (Kv) juguen un paper molt important tant en cèl•lules excitables com no excitables. La possibilitat de formar hetero-oligomers i la d’associació amb subunitats reguladores són uns dels mecanismes que existeixen per tal de proveir de diferents mecanismes per a respondre de manera diferent enfront a canvis en el potencial de membrana. La composició del canalosoma modula tant la seva expressió a superfície com l’activitat d’aquests. Aquesta tesi es centra en l’estudi de l’efecte del la família de les subunitats reguladores KCNE sobre diferents Kv. Primerament s’estudià l’efecte que causaven en el tràfic del canal Kv7.1 (canal model per a l’estudi dels KCNEs) i posteriorment s’amplià l’estudi a un altre membre de la mateixa família, Kv7.5. A continuació s’estudià un canal d’elevada importància per a l’activació i proliferació leucocitària: Kv1.3, centrant-nos sobretot en l’efecte causat per un dels KCNEs: KCNE4. Aquesta subunitat no només inhibeix dràsticament el corrent del canal Kv1.3, sinó que a més a més, modifica el seu tràfic i localització. Aquests canvis són deguts a una interacció directa entre ambdues proteïnes. A continuació s’estudià en detall el complex Kv1.3-KCNE4. Mitjançant la combinació d’experiments d’electrofisiologia i monitorització de fluorescència de molècules individuals en la membrana, es va poder establir l’estequiometria d’aquest complex. Posteriorment, mitjançant l’anàlisi de diverses proteïnes quimèriques i mutants, tant del canal com de la subunitat reguladora, es van cercar els determinants moleculars implicats en l’associació entre ambdues proteïnes. S’han pogut determinar els motius claus en KCNE4 i Kv1.3 implicats en la formació del complex, però no en la modulació del canal. Finalment, degut a la importància de Kv1.3 en el sistema immunitari, s’han analitzat els nivells d’expressió dels KCNEs en diferents línies leucocitàries. S’ha observat que aquestes subunitats pateixen una regulació diferencial en funció de la manera d’activació i al llarg de la proliferació del leucòcits, suggerint un possible paper en la regulació precisa de la resposta immunològica
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the kcne2 potassium channel β subunit is required for normal lung function and resilience to ischemia and reperfusion injury
The FASEB Journal, 2019Co-Authors: Leng Zhou, Torsten K Roepke, Clemens Kohncke, Geoffrey W AbbottAbstract:The KCNE2 single transmembrane-spanning voltage-gated potassium (Kv) channel β subunit is ubiquitously expressed and essential for normal function of a variety of cell types, often via regulation of the KCNQ1 Kv channel. A polymorphism upstream of KCNE2 is associated with reduced lung function in human populations, but the pulmonary consequences of KCNE2 gene disruption are unknown. Here, germline deletion of mouse Kcne2 reduced pulmonary expression of potassium channel α subunits Kcnq1 and Kcnb1 but did not alter expression of other Kcne genes. Kcne2 colocalized and coimmunoprecipitated with Kcnq1 in mouse lungs, suggesting the formation of pulmonary Kcnq1-Kcne2 potassium channel complexes. Kcne2 deletion reduced blood O2, increased CO2, increased pulmonary apoptosis, and increased inflammatory mediators TNF-α, IL-6, and leukocytes in bronchoalveolar lavage (BAL) fluids. Consistent with increased pulmonary vascular leakage, Kcne2 deletion increased plasma, BAL albumin, and the BAL:plasma albumin concentration ratio. Kcne2-/- mouse lungs exhibited baseline induction of the reperfusion injury salvage kinase pathway but were less able to respond via this pathway to imposed pulmonary ischemia/reperfusion injury (IRI). We conclude that KCNE2 regulates KCNQ1 in the lungs and is required for normal lung function and resistance to pulmonary IRI. Our data support a causal relationship between KCNE2 gene disruption and lung dysfunction.-Zhou, L., Kohncke, C., Hu, Z., Roepke, T. K., Abbott, G. W. The KCNE2 potassium channel β subunit is required for normal lung function and resilience to ischemia and reperfusion injury.
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effects of kcne subunit deletion on polarized trafficking of the kcnq1 potassium channel in vivo
Biophysical Journal, 2010Co-Authors: Geoffrey W Abbott, Kerry Purtell, Elizabeth C King, Gianina Panaghie, Daniel Lerner, Torsten K RoepkeAbstract:The KCNQ1 potassium channel alpha subunit generates essential K+ currents in human heart and in a range of polarized secretory epithelia. The polarity of KCNQ1 trafficking varies between different epithelia, but neither the importance nor the mechanism for this polarity are well understood. KCNQ1 co-localizes apically with the KCNE2 beta subunit in gastric parietal cells but basolaterally with KCNE3 in colonic crypts. Both KCNE2 and KCNE3 convert KCNQ1 to a constitutively active channel. Here, genetic deletion of Kcne2 in mice resulted in 5-fold upregulation of KCNE3, formation of Kcnq1-KCNE3 complexes, and basolateral Kcnq1 targeting in parietal cells, and gastritis cystica profunda stemming from achlorhydria and earlier hyperplasia. In contrast, Kcne2-/-KCNE3-/- mice exhibited apical parietal cell Kcnq1 localization. Thus, in parietal cells, apical Kcnq1 localization is required for gastric acid secretion, and the apical localization per se does not require Kcne2. KCNE3, if present, actively targets Kcnq1 basolaterally, ultimately causing a preneoplastic condition which in humans could predispose to gastric cancer.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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the kcne2 potassium channel ancillary subunit is essential for gastric acid secretion
Journal of Biological Chemistry, 2006Co-Authors: Torsten K Roepke, Philipp Kirchhoff, Jeffrey B Young, Daniel J Lerner, John P. Geibel, Stephanie M Busque, Arun Anantharam, Geoffrey W AbbottAbstract:Abstract Genes in the KCNE family encode single transmembrane domain ancillary subunits that co-assemble with voltage-gated potassium (Kv) channel α subunits to alter their function. KCNE2 (also known as MiRP1) is expressed in the heart, is associated with human cardiac arrhythmia, and modulates cardiac Kv α subunits hERG and KCNQ1 in vitro. KCNE2 and KCNQ1 are also expressed in parietal cells, leading to speculation they form a native channel complex there. Here, we disrupted the murine kcne2 gene and found that kcne2 (-/-) mice have a severe gastric phenotype with profoundly reduced parietal cell proton secretion, abnormal parietal cell morphology, achlorhydria, hypergastrinemia, and striking gastric glandular hyperplasia arising from an increase in the number of non-acid secretory cells. KCNQ1 exhibited abnormal distribution in gastric glands from kcne2 (-/-) mice, with increased expression in non-acid secretory cells. Parietal cells from kcne2 (+/-) mice exhibited normal architecture but reduced proton secretion, and kcne2 (+/-) mice were hypochlorhydric, indicating a gene-dose effect and a primary defect in gastric acid secretion. These data demonstrate that KCNE2 is essential for gastric acid secretion, the first genetic evidence that a member of the KCNE gene family is required for normal gastrointestinal function.