| 1. |
- 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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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
- Pantazis, Antonios, 1982-
(författare)
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Cut-open Oocyte Voltage Clamp Technique
- 2013. - 1
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Ingår i: Encyclopedia of Biophysics. - : Springer Berlin/Heidelberg. - 9783642167119 - 9783642167133 - 9783642167126 ; , s. 406-413
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The cut-open oocyte Vaseline gap (COVG) voltage-clamp technique, a relatively recent addition to theelectrophysiologist’s armamentarium, was specificallydeveloped by Drs. Stefani and Bezanilla (Bezanillaet al.1991) to achieve low-noise recordings of themembrane ofXenopus laevisoocytes with fast clampspeed and, thus, optimize the most popular transientexpression system to reveal the activity voltage-dependent proteins previously difficult to resolve byalternative methods. The high degree of specializationof this technique is complemented by its flexibility: inaddition to oocyte perfusion, COVG can beencombined with optical measurements (voltage-clamp fluorometry and spectroscopy) and flash pho-tolysisfor the instantaneous release of intracellular-caged compounds, expanding its use beyondelectrophysiology.
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| 6. |
- Pantazis, Antonios, 1982-, et al.
(författare)
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Cut-Open Oocyte Voltage-Clamp Technique
- 2019. - Living Edition
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Ingår i: Encyclopedia of Biophysics. - Berlin, Heidelberg : Springer Berlin/Heidelberg. - 9783642359439
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Bokkapitel (refereegranskat)abstract
- The cut-open oocyte Vaseline gap (COVG) voltage clamp technique, a relatively recent addition to the electrophysiologist’s armamentarium, was specifically developed by Drs. Stefani and Bezanilla (Bezanilla et al. 1991) to achieve low-noise recordings of the membrane of Xenopus laevis oocytes with fast clamp speed and thus optimize the most popular transient expression system to reveal the activity voltage-dependent proteins previously difficult to resolve by alternative methods. The high degree of specialization of this technique is complemented by its flexibility; in addition to oocyte perfusion, COVG can be combined with optical measurements (voltage clamp fluorometry and spectroscopy) and flash photolysis for the instantaneous release of intracellular caged compounds, expanding its use beyond electrophysiology.
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| 7. |
- Pantazis, Antonios, 1982-, et al.
(författare)
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Distinct Roles for Two Histamine Receptors (hclA and hclB) at the Drosophila Photoreceptor Synapse
- 2008
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Ingår i: Journal of Neuroscience. - : Society for Neuroscience. - 0270-6474 .- 1529-2401. ; 28:29, s. 7250-7259
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Tidskriftsartikel (refereegranskat)abstract
- Histamine (HA) is the photoreceptor neurotransmitter in arthropods, directly gating chloride channels on large monopolar cells (LMCs), postsynaptic to photoreceptors in the lamina. Two histamine-gated channel genes that could contribute to this channel in Drosophila are hclA (also known as ort) and hclB (also known as hisCl1), both encoding novel members of the Cys-loop receptor superfamily. Drosophila S2 cells transfected with these genes expressed both homomeric and heteromeric histamine-gated chloride channels. The electrophysiological properties of these channels were compared with those from isolated Drosophila LMCs. HCLA homomers had nearly identical HA sensitivity to the native receptors (EC50 = 25 ÎŒm). Single-channel analysis revealed further close similarity in terms of single-channel kinetics and subconductance states (~25, 40, and 60 pS, the latter strongly voltage dependent). In contrast, HCLB homomers and heteromeric receptors were more sensitive to HA (EC50 = 14 and 1.2 ÎŒm, respectively), with much smaller single-channel conductances (~4 pS). Null mutations of hclA (ortUS6096) abolished the synaptic transients in the electroretinograms (ERGs). Surprisingly, the ERG “on” transients in hclB mutants transients were approximately twofold enhanced, whereas intracellular recordings from their LMCs revealed altered responses with slower kinetics. However, HCLB expression within the lamina, assessed by both a GFP (green fluorescent protein) reporter gene strategy and mRNA tagging, was exclusively localized to the glia cells, whereas HCLA expression was confirmed in the LMCs. Our results suggest that the native receptor at the LMC synapse is an HCLA homomer, whereas HCLB signaling via the lamina glia plays a previously unrecognized role in shaping the LMC postsynaptic response.
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| 8. |
- Pantazis, Antonios, 1982-, et al.
(författare)
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Relative Motion of Transmembrane Segments S0 and S4 during Voltage Sensor Activation in the Human BKCa Channel
- 2010
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Ingår i: The Journal of General Physiology. - : Rockefeller University Press. - 0022-1295 .- 1540-7748. ; 136:6, s. 645-657
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Tidskriftsartikel (refereegranskat)abstract
- Large-conductance voltage- and Ca2+-activated K+ (BKCa) channel α subunits possess a unique transmembrane helix referred to as S0 at their N terminus, which is absent in other members of the voltage-gated channel superfamily. Recently, S0 was found to pack close to transmembrane segments S3 and S4, which are important components of the BKCa voltage-sensing apparatus. To assess the role of S0 in voltage sensitivity, we optically tracked protein conformational rearrangements from its extracellular flank by site-specific labeling with an environment-sensitive fluorophore, tetramethylrhodamine maleimide (TMRM). The structural transitions resolved from the S0 region exhibited voltage dependence similar to that of charge-bearing transmembrane domains S2 and S4. The molecular determinant of the fluorescence changes was identified in W203 at the extracellular tip of S4: at hyperpolarized potential, W203 quenches the fluorescence of TMRM labeling positions at the N-terminal flank of S0. We provide evidence that upon depolarization, W203 (in S4) moves away from the extracellular region of S0, lifting its quenching effect on TMRM fluorescence. We suggest that S0 acts as a pivot component against which the voltage-sensitive S4 moves upon depolarization to facilitate channel activation.
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| 9. |
- Pantazis, Antonios, 1982-, et al.
(författare)
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The Effect of Neuronal Morphology and Membrane-permeant Weak Acid and Base on the Dissipation of Depolarization-induced pH Gradients in Snail Neurons
- 2006
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Ingår i: Pflügers Archiv. - : Springer. - 0031-6768 .- 1432-2013. ; 452:2, s. 175-187
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Tidskriftsartikel (refereegranskat)abstract
- Neuronal depolarization causes larger intracellular pH (pHi) shifts in axonal and dendritic regions than in the cell body. In this paper, we present evidence relating the time for collapse of these gradients to neuronal morphology. We have used ratiometric pHi measurements using 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) in whole-cell patch-clamped snail neurons to study the collapse of longitudinal pH gradients. Using depolarization to open voltage-gated proton channels, we produced alkaline pHi microdomains. In the absence of added mobile buffers, facilitated H+ diffusion down the length of the axon plays a critical role in determining pHi microdomain lifetime, with axons of ∼100 μm allowing pH differences to be maintained for >60 s. An application of mobile, membrane-permeant pH buffers accelerated the collapse of the alkaline-pH gradients but, even at 30 mM, was unable to abolish them. Modeling of the pHi dynamics showed that both the relatively weak effect of the weak acid/base on the peak size of the pH gradient and the accelerated collapse of the pH gradient could be due to the time taken for equilibration of the weak acid and base across the cell. We propose that appropriate weak acid/base mixes may provide a simple method for studying the role of local pHi signals without perturbing steady-state pHi. Furthermore, an extrapolation of our in vitro data to longer and thinner neuronal structures found in the mammalian nervous system suggests that dendritic and axonal pHi are likely to be dominated by local pHi-regulating mechanisms rather than simply following the soma pHi.
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| 10. |
- Savalli, Nicoletta, et al.
(författare)
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The contribution of RCK domains to human BK channel allosteric activation
- 2012
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 287:26, s. 21741-21750
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Tidskriftsartikel (refereegranskat)abstract
- Large conductance voltage- and Ca2+-activated K+ (BK) channels are potent regulators of cellular processes including neuronal firing, synaptic transmission, cochlear hair cell tuning, insulin release, and smooth muscle tone. Their unique activation pathway relies on structurally distinct regulatory domains including one transmembrane voltage-sensing domain (VSD) and two intracellular high affinity Ca2+-sensing sites per subunit (located in the RCK1 and RCK2 domains). Four pairs of RCK1 and RCK2 domains form a Ca2+-sensing apparatus known as the “gating ring.” The allosteric interplay between voltage- and Ca2+-sensing apparati is a fundamental mechanism of BK channel function. Using voltage-clamp fluorometry and UV photolysis of intracellular caged Ca2+, we optically resolved VSD activation prompted by Ca2+ binding to the gating ring. The sudden increase of intracellular Ca2+ concentration ([Ca2+]i) induced a hyperpolarizing shift in the voltage dependence of both channel opening and VSD activation, reported by a fluorophore labeling position 202, located in the upper side of the S4 transmembrane segment. The neutralization of the Ca2+ sensor located in the RCK2 domain abolished the effect of [Ca2+]i increase on the VSD rearrangements. On the other hand, the mutation of RCK1 residues involved in Ca2+ sensing did not prevent the effect of Ca2+ release on the VSD, revealing a functionally distinct interaction between RCK1 and RCK2 and the VSD. A statistical-mechanical model quantifies the complex thermodynamics interplay between Ca2+ association in two distinct sites, voltage sensor activation, and BK channel opening.
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