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A V-to-F Substitution in SK2 Channels Causes Ca2+ Hypersensitivity and Improves Locomotion in a C. elegans ALS Model

Published on Aug 1, 2018in Scientific Reports4.011
· DOI :10.1038/s41598-018-28783-2
Young-Woo Nam4
Estimated H-index: 4
(Chapman University),
Sabia N. Baskoylu1
Estimated H-index: 1
(Brown University)
+ 4 AuthorsMiao Zhang7
Estimated H-index: 7
(Chapman University)
Sources
Abstract
Small-conductance Ca2+-activated K+ (SK) channels mediate medium afterhyperpolarization in the neurons and play a key role in the regulation of neuronal excitability. SK channels are potential drug targets for ataxia and Amyotrophic Lateral Sclerosis (ALS). SK channels are activated exclusively by the Ca2+-bound calmodulin. Previously, we identified an intrinsically disordered fragment that is essential for the mechanical coupling between Ca2+/calmodulin binding and channel opening. Here, we report that substitution of a valine to phenylalanine (V407F) in the intrinsically disordered fragment caused a ~6 fold increase in the Ca2+ sensitivity of SK2-a channels. This substitution resulted in a novel interaction between the ectopic phenylalanine and M411, which stabilized PIP2-interacting residue K405, and subsequently enhanced Ca2+ sensitivity. Also, equivalent valine to phenylalanine substitutions in SK1 or SK3 channels conferred Ca2+ hypersensitivity. An equivalent phenylalanine substitution in the Caenorhabditis elegans (C. elegans) SK2 ortholog kcnl-2 partially rescued locomotion defects in an existing C. elegans ALS model, in which human SOD1G85R is expressed at high levels in neurons, confirming that this phenylalanine substitution impacts channel function in vivo. This work for the first time provides a critical reagent for future studies: an SK channel that is hypersensitive to Ca2+ with increased activity in vivo.
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  • Citations (1)
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References46
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#1Chia-Hsueh Lee (HHMI: Howard Hughes Medical Institute)H-Index: 2
#2Roderick MacKinnon (HHMI: Howard Hughes Medical Institute)H-Index: 88
Small-conductance Ca 2+ -activated K + (SK) channels mediate neuron excitability and are associated with synaptic transmission and plasticity. They also regulate immune responses and the size of blood cells. Activation of SK channels requires calmodulin (CaM), but how CaM binds and opens SK channels has been unclear. Here we report cryo–electron microscopy (cryo-EM) structures of a human SK4-CaM channel complex in closed and activated states at 3.4- and 3.5-angstrom resolution, respectively. Fou...
21 CitationsSource
#1Young Woo Nam (Chapman University)H-Index: 1
#2Razan Orfali (Chapman University)H-Index: 2
Last. Miao Zhang (Chapman University)H-Index: 7
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Small-conductance Ca2+-activated K+ (SK) channels play essential roles in the regulation of cellular excitability and have been implicated in neurological and cardiovascular diseases through both animal model studies and human genetic association studies. Over the past two decades, positive modulators of SK channels such as NS309 and 1-EBIO have been developed. Our previous structural studies have identified the binding pocket of 1-EBIO and NS309 that is located at the interface between the chan...
4 CitationsSource
#1Polina A. Egorova (Saint Petersburg State Polytechnic University)H-Index: 6
#2O. A. Zakharova (Saint Petersburg State Polytechnic University)H-Index: 4
Last. Ilya Bezprozvanny (University of Texas at Austin)H-Index: 59
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Cerebellar Purkinje cells (PCs) are primarily affected in many spinocerebellar ataxias (SCA). In this study we investigated functional activity of PCs in transgenic mouse model of SCA2, a polyglutamine neurodegenerative hereditary disorder. In our studies we used extracellular single-unit recording method to compare spontaneous activity of PCs in age-matched wild-type mice and SCA2-58Q transgenic mice. We discovered that the fraction of PCs with bursting and an irregular pattern of spontaneous a...
23 CitationsSource
#1Silvia Romano (Sapienza University of Rome)H-Index: 18
#2Giulia Coarelli (Sapienza University of Rome)H-Index: 10
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Summary Background Our previous study in patients with cerebellar ataxias of different causes showed significant benefit of riluzole after 8 weeks. We aimed to confirm these results in patients with spinocerebellar ataxia or Friedreich's ataxia in a 1-year trial. Methods Patients with spinocerebellar ataxia or Friedreich's ataxia (2:1 ratio) from three Italian neurogenetic units were enrolled in this multicentre, double-blind, placebo-controlled trial, and randomly assigned to riluzole (50 mg or...
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#1Miao Zhang (VCU: Virginia Commonwealth University)H-Index: 7
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Phosphatidylinositol 4,5-bisphosphate (PIP2) directly interacts with the small-conductance Ca2+-activated K+ 2-a (SK2-a) channel/calmodulin complex, serving as a critical element in the regulation of channel activity. We report that changes of protein conformation in close proximity to the PIP2 binding site induced by a small-molecule SK channel modulator, NS309, can effectively enhance the interaction between the protein and PIP2 to potentiate channel activity. This novel modulation of PIP2 sen...
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#1Jordan D. Ward (UCSF: University of California, San Francisco)H-Index: 20
As in other organisms, CRISPR/Cas9 methods provide a powerful approach for genome editing in the nematode Caenorhabditis elegans. Oligonucleotides are excellent repair templates for introducing substitutions and short insertions, as they are cost effective, require no cloning, and appear in other organisms to target changes by homologous recombination at DNA double-strand breaks (DSBs). Here, I describe a methodology in C. elegans to efficiently knock in epitope tags in 8–9 days, using a tempera...
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#1Miao ZhangH-Index: 7
#2Xuan-Yu MengH-Index: 11
Last. Ji-fang ZhangH-Index: 22
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SK potassium channels are activated by Ca2+-bound calmodulin (CaM) and regulated by phosphorylation. Electrophysiology and MD simulations show that a PIP2-binding site formed at the interface of CaM and SK2, conferring PIP2 sensitivity on the channels.
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#1Katiuska Luna-Cancalon (UM: University of Michigan)H-Index: 3
#2Kristine M. Sikora (UM: University of Michigan)H-Index: 2
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Recent evidence suggests that dystonia, a movement disorder characterized by sustained involuntary muscle contractions, can be associated with cerebellar abnormalities. The basis for how functional changes in the cerebellum can cause dystonia is poorly understood. Here we identify alterations in physiology in Atcayji-hes mice which in addition to ataxia, have an abnormal gait with hind limb extension and toe walking, reminiscent of human dystonic gait. No morphological abnormalities in the brain...
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#1Elizabeth S torer Scholl (Tufts University)H-Index: 1
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Small conductance Ca2+-sensitive potassium (SK2) channels are voltage-independent, Ca2+-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca2+ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective...
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#1Cavita K. Chotoo (University of Pittsburgh)H-Index: 3
#2Gary A. Silverman (University of Pittsburgh)H-Index: 37
Last. Cliff J. Luke (University of Pittsburgh)H-Index: 22
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In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson’s disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK chan...
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Intracellular calcium is essential for many physiological processes, from neuronal signaling and exocytosis to muscle contraction and bone formation. Ca2+ signaling from the extracellular medium depends both on membrane potential, especially controlled by ion channels selective to K+, and direct permeation of this cation through specialized channels. Calmodulin (CaM), through direct binding to these proteins, participates in setting the membrane potential and the overall permeability to Ca2+. Ov...
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#1Christiane K. Bauer (UHH: University of Hamburg)H-Index: 23
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Zimmermann-Laband syndrome (ZLS) is characterized by coarse facial features with gingival enlargement, intellectual disability (ID), hypertrichosis, and hypoplasia or aplasia of nails and terminal phalanges. De novo missense mutations in KCNH1 and KCNK4, encoding K+ channels, have been identified in subjects with ZLS and ZLS-like phenotype, respectively. We report de novo missense variants in KCNN3 in three individuals with typical clinical features of ZLS. KCNN3 (SK3/KCa2.3) constitutes one of ...
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