| 1. |
- Angelini, Marina, et al.
(författare)
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Suppression of ventricular arrhythmias by targeting late L-type Ca2+ current
- 2021
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Ingår i: The Journal of General Physiology. - : ROCKEFELLER UNIV PRESS. - 0022-1295 .- 1540-7748. ; 153:12
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Tidskriftsartikel (refereegranskat)abstract
- Ventricular arrhythmias, a leading cause of sudden cardiac death, can be triggered by cardiomyocyte early afterdepolarizations (EADs). EADs can result from an abnormal late activation of L-type Ca2+ channels (LTCCs). Current LTCC blockers (class IV antiarrhythmics), while effective at suppressing EADs, block both early and late components of I-Ca,I-L, compromising inotropy. However, computational studies have recently demonstrated that selective reduction of late I-Ca,I-L (Ca2+ influx during late phases of the action potential) is sufficient to potently suppress EADs, suggesting that effective antiarrhythmic action can be achieved without blocking the early peak I-Ca,I-L, which is essential for proper excitation-contraction coupling. We tested this new strategy using a purine analogue, roscovitine, which reduces late I-Ca,I-L with minimal effect on peak current. Scaling our investigation from a human Ca(V)1.2 channel clone to rabbit ventricular myocytes and rat and rabbit perfused hearts, we demonstrate that (1) roscovitine selectively reduces I-Ca,I-L noninactivating component in a human Ca(V)1.2 channel clone and in ventricular myocytes native current, (2) the pharmacological reduction of late I-Ca,I-L suppresses EADs and EATs (early after Ca2+ transients) induced by oxidative stress and hypokalemia in isolated myocytes, largely preserving cell shortening and normal Ca2+ transient, and (3) late I-Ca,I-L reduction prevents/suppresses ventricular tachycardia/fibrillation in ex vivo rabbit and rat hearts subjected to hypokalemia and/or oxidative stress. These results support the value of an antiarrhythmic strategy based on the selective reduction of late I-Ca,I-L to suppress EAD-mediated arrhythmias. Antiarrhythmic therapies based on this idea would modify the gating properties of Ca(V)1.2 channels rather than blocking their pore, largely preserving contractility.
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| 2. |
- Hoshi, T., et al.
(författare)
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Transduction of Voltage and Ca2+ Signals by Slo1 BK Channels
- 2013
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Ingår i: Physiology (Bethesda). - : Amercian Physiolocial Society. - 1548-9213 .- 1548-9221. ; 28:3, s. 172-189
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Forskningsöversikt (refereegranskat)abstract
- Large-conductance Ca2+- and voltage-gated K+ channels are activated by an increase in intracellular Ca2+ concentration and/or depolarization. The channel activation mechanism is well described by an allosteric model encompassing the gate, voltage sensors, and Ca2+ sensors, and the model is an excellent framework to understand the influences of auxiliary β and γ subunits and regulatory factors such as Mg2+. Recent advances permit elucidation of structural correlates of the biophysical mechanism.
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| 3. |
- Javaherian, Anoosh D., et al.
(författare)
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Metal-driven operation of the human large-conductance voltage- and Ca2+-dependent potassium channel (BK) gating ring apparatus
- 2011
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 286:23, s. 20701-20709
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Tidskriftsartikel (refereegranskat)abstract
- Large-conductance voltage- and Ca2+-dependent K+ (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca2+ homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the “gating ring”), which confers sensitivity to intracellular Ca2+ and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca2+- and Mg2+-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba2+-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca2+-binding site (the “calcium bowl”) reduced the Ca2+ and abolished the Mg2+ dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca2+-binding site and that Mg2+ can bind to the calcium bowl with less affinity than Ca2+. Dynamic light scattering analysis revealed a reversible Ca2+-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.
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| 4. |
- Madhvani, Roshni V., et al.
(författare)
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Shaping a New Ca2+ Conductance to Suppress Early Afterdepolarizations in Cardiac Myocytes
- 2011
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Ingår i: Journal of Physiology. - : John Wiley & Sons. - 0022-3751 .- 1469-7793. ; 589:24, s. 6081-6092
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Tidskriftsartikel (refereegranskat)abstract
- Non‐technical summary Diseases, genetic defects, or ionic imbalances can alter the normal electrical activity of cardiac myocytes causing an anomalous heart rhythm, which can degenerate to ventricular fibrillation (VF) and sudden cardiac death. Well‐recognized triggers for VF are aberrations of the cardiac action potential, known as early afterdepolarizations (EADs). In this study, combining mathematical modelling and experimental electrophysiology in real‐time (dynamic clamp), we investigated the dependence of EADs on the biophysical properties of the L‐type Ca2+ current (ICa,L) and identified modifications of ICa,L properties which effectively suppress EAD. We found that minimal changes in the voltage dependence of activation or inactivation of ICa,L can dramatically reduce the occurrence of EADs in cardiac myocytes exposed to different EAD‐inducing conditions. This work assigns a critical role to the L‐type Ca2+ channel biophysical properties for EADs formation and identifies the L‐type Ca2+ channel as a promising therapeutic target to suppress EADs and their arrhythmogenic effects.
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| 5. |
- Madhvani, Roshni V., et al.
(författare)
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Targeting the Late Component of the Cardiac L-type Ca2+ Current to Suppress Early Afterdepolarizations
- 2015
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Ingår i: The Journal of General Physiology. - : Rockefeller University Press. - 0022-1295 .- 1540-7748. ; 145:5, s. 395-404
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Tidskriftsartikel (refereegranskat)abstract
- Early afterdepolarizations (EADs) associated with prolongation of the cardiac action potential (AP) can create heterogeneity of repolarization and premature extrasystoles, triggering focal and reentrant arrhythmias. Because the L-type Ca2+ current (ICa,L) plays a key role in both AP prolongation and EAD formation, L-type Ca2+ channels (LTCCs) represent a promising therapeutic target to normalize AP duration (APD) and suppress EADs and their arrhythmogenic consequences. We used the dynamic-clamp technique to systematically explore how the biophysical properties of LTCCs could be modified to normalize APD and suppress EADs without impairing excitation–contraction coupling. Isolated rabbit ventricular myocytes were first exposed to H2O2 or moderate hypokalemia to induce EADs, after which their endogenous ICa,L was replaced by a virtual ICa,L with tunable parameters, in dynamic-clamp mode. We probed the sensitivity of EADs to changes in the (a) amplitude of the noninactivating pedestal current; (b) slope of voltage-dependent activation; (c) slope of voltage-dependent inactivation; (d) time constant of voltage-dependent activation; and (e) time constant of voltage-dependent inactivation. We found that reducing the amplitude of the noninactivating pedestal component of ICa,L effectively suppressed both H2O2- and hypokalemia-induced EADs and restored APD. These results, together with our previous work, demonstrate the potential of this hybrid experimental–computational approach to guide drug discovery or gene therapy strategies by identifying and targeting selective properties of LTCC.
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| 6. |
- Mínguez‐Viñas, Teresa, et al.
(författare)
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Two epilepsy‐associated variants in KCNA2 (KV1.2) at position H310 oppositely affect channel functional expression
- 2023
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Ingår i: Journal of Physiology. - : WILEY. - 0022-3751 .- 1469-7793. ; 601:23, s. 5367-5389
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Tidskriftsartikel (refereegranskat)abstract
- Two KCNA2 variants (p.H310Y and p.H310R) were discovered in paediatric patients with epilepsy and developmental delay. KCNA2 encodes KV1.2-channel subunits, which regulate neuronal excitability. Both gain and loss of KV1.2 function cause epilepsy, precluding the prediction of variant effects; and while H310 is conserved throughout the KV-channel superfamily, it is largely understudied. We investigated both variants in heterologously expressed, human KV1.2 channels by immunocytochemistry, electrophysiology and voltage-clamp fluorometry. Despite affecting the same channel, at the same position, and being associated with severe neurological disease, the two variants had diametrically opposite effects on KV1.2 functional expression. The p.H310Y variant produced ‘dual gain of function’, increasing both cell-surface trafficking and activity, delaying channel closure. We found that the latter is due to the formation of a hydrogen bond that stabilizes the active state of the voltage-sensor domain. Additionally, H310Y abolished ‘ball and chain’ inactivation of KV1.2 by KVβ1 subunits, enhancing gain of function. In contrast, p.H310R caused ‘dual loss of function’, diminishing surface levels by multiple impediments to trafficking and inhibiting voltage-dependent channel opening. We discuss the implications for KV-channel biogenesis and function, an emergent hotspot for disease-associated variants, and mechanisms of epileptogenesis.
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| 7. |
- Nilsson, Michelle, et al.
(författare)
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An epilepsy-associated KV1.2 charge-transfer-center mutation impairs KV1.2 and KV1.4 trafficking
- 2022
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 119:17
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Tidskriftsartikel (refereegranskat)abstract
- Significance: A child with epilepsy has a previously unreported, heterozygous mutation in KCNA2, the gene encoding KV1.2 proteins. Four KV1.2 assemble into a potassium-selective channel, a protein complex at the neuronal cell surface regulating electrical signaling. KV1.2 subunits assemble with other KV1-family members to form heterotetrameric channels, contributing to neuronal potassium-channel diversity. The most striking consequence of this mutation is preventing KV1.2-subunit trafficking, i.e., their ability to reach the cell surface. Moreover, the mutation is dominant negative, as mutant subunits can assemble with wild-type KV1.2 and KV1.4, trapping them into nontrafficking heterotetramers and decreasing their functional expression. Thus, KV1-family genes’ ability to form heterotetrameric channels is a double-edged sword, rendering KV1-family members vulnerable to dominant-negative mutations in a single member gene.Abstract: We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2 or KV1.4, respectively, and that both early and late events along the biosynthesis and secretion pathway impair trafficking. These studies suggested that F233S causes a depolarizing shift of ∼48 mV on KV1.2 voltage dependence. Optical tracking of the KV1.2(F233S) voltage-sensing domain (rescued by wild-type KV1.2 or KV1.4) revealed that it operates with modestly perturbed voltage dependence and retains pore coupling, evidenced by off-charge immobilization. The equivalent mutation in the Shaker K+ channel (F290S) was reported to modestly affect trafficking and strongly affect function: an ∼80-mV depolarizing shift, disrupted voltage sensor activation and pore coupling. Our work exposes the multigenic, molecular etiology of a variant associated with epilepsy and reveals that charge-transfer-center disruption has different effects in KV1.2 and Shaker, the archetypes for potassium channel structure and function.
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| 8. |
- Nilsson, Michelle, 1993-
(författare)
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Voltage-Sensor Domains of Ion Channels : Physiology, Regulation, and Role in Disease
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Brain function depends on the ability of neurons to sense and respond to electricity, which is mediated by small modules in the neuronal membrane called voltage-sensor domains (VSDs). Disruption of VSD function can cause neurological disease such as epilepsy. VSDs contain positively charged amino acids that move in response to changes in membrane potential. This movement transfer energy to other coupled effectors, such as the pore of a voltage-gated ion channel. In this thesis, I have studied the physiology and regulation of ion-channel VSDs, as well as their role in disease.Voltage-gated ion channels are composed of four VSDs that controls the opening of a central ion-conducting pore. Voltage-gated potassium (KV) channels are tetramers assembled by four subunits, where each subunit consists of a VSD and 1/4 of the pore. In contrast, voltage-gated sodium (NaV) and voltage-gated calcium (CaV) channels are pseudotetramers composed of four non-identical, concatenated subunits (repeats I-IV). Our genes encode a broad repertoire of voltage-gated ion channels, promoting diversity and specialization of neuronal subtypes. Specifically, 40 KV-, 9 NaV-, and 10 CaV-channels have been identified. This thesis includes studies on i) VSD operation in the CaV2.2 channel, known for its role in pain transmission, ii) G-proteins Gβγ inhibition of CaV2.2 VSDs, a potential tool to control pain, and iii) characterization of two different epilepsy-associated mutations in the VSD of the KV1.2 channel, important for repolarization of the action potential. To do this, the methods voltage-clamp fluorometry (VCF) under cut-open oocyte voltage clamp mode using Xenopus oocytes, or flow cytometry using a mammalian cell line (COS-7) were used.VCF was implemented in the human CaV2.2 channel and VSD activation in relation to pore opening was characterized. The voltage dependence of VSD-I activation was found to correlate with pore opening, VSD II is likely immobile (it did not generate any VCF signals), VSD III activated at very negative potentials, and VSD IV activation had similar voltagedependence to that of pore opening. Next, Gβγ-inhibition of the VSDs was explored. VSD I was strongly and proportionally inhibited compared to pore opening, VSD III was unaffected and VSD IV was modestly inhibited. In the following studies, the role of the KV1.2-VSD in disease was explored. Two different epilepsy-associated mutations in the VSD of KV1.2 were characterized. The first mutation, F302L, facilitated channel activation and spontaneous closure (inactivation) without affecting surface trafficking. The second mutation, F233S, caused a severe surface trafficking deficiency, extending to WT-subunits and closely related KV1.4 partner subunits. In conclusion, VSDs of ion channels are fundamental for the complexity of our nervous system, their regulation can be used to further diversify neurons or to control excitability, and their importance is revealed by disease-associated mutations that prevent normal function.
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| 9. |
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| 10. |
- Pantazis, Antonios, et al.
(författare)
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Biophysics of BK Channel Gating
- 2016
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Ingår i: International review of neurobiology. - : Elsevier. - 0074-7742 .- 2162-5514. ; 128, s. 1-49
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Forskningsöversikt (refereegranskat)abstract
- BK channels are universal regulators of cell excitability, given their exceptional unitary conductance selective for K(+), joint activation mechanism by membrane depolarization and intracellular [Ca(2+)] elevation, and broad expression pattern. In this chapter, we discuss the structural basis and operational principles of their activation, or gating, by membrane potential and calcium. We also discuss how the two activation mechanisms interact to culminate in channel opening. As members of the voltage-gated potassium channel superfamily, BK channels are discussed in the context of archetypal family members, in terms of similarities that help us understand their function, but also seminal structural and biophysical differences that confer unique functional properties.
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