Activation mechanism of a human SK-calmodulin channel complex elucidated by cryo-EM structures

Lee, Chia‐Hsueh; MacKinnon, Roderick

doi:10.1126/science.aas9466

Cited by 144 publications

(249 citation statements)

References 78 publications

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“…Moreover,t he radiationless deactivation rates are drastically higher for the latter and significantly enhanced for 19 and 20,ifcompared with 16-18.This is surprising since [16][17][18] have additional side chains providing extra degrees of freedom and therefore ah igher density of rotovibrational states that could favor non-radiative relaxation pathways.T his points to radiationless transitions that are mostly governed by electronic effects associated to the variable substitution patterns.T he latter is evident for 19-20 but particularly for 15,ascan be also observed in the more polar CH 2 Cl 2 /CH 3 OH mixture in which all cases the radiative relaxation rates are (modified accordingto literature [13] ). slightly slower than in CH 3 CN while the radiationless processes are somewhat faster than in the less polar environment (Table 3).…”

Section: Synthesis Of the Probesmentioning

confidence: 98%

Synthesis of Small‐Molecule Fluorescent Probes for the In Vitro Imaging of Calcium‐Activated Potassium Channel K_Ca3.1

et al. 2020

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Small-molecule probes for the in vitro imaging of K Ca 3.1 channel-expressing cells were developed. Senicapoc, showing high affinity and selectivity for the K Ca 3.1 channels, was chosen as the targeting component. BODIPY dyes 15-20 were synthesized and connected by aC u I -catalyzed azidealkyne [3+ +2]cycloaddition with propargyl ether senicapoc derivative 8,yielding fluorescently labeled ligands 21-26.The dimethylpyrrole-based imaging probes 25 and 26 allow staining of K Ca 3.1 channels in NSCLC cells.T he specificity was shown by removing the punctate staining pattern by preincubation with senicapoc.T he density of K Ca 3.1 channels detected with 25 and by immunostaining was identical. The punctate structure of the labeled channels could also be observed in living cells.M olecular modeling showed binding of the senicapoc-targeting component towards the binding site within the ion channel and orientation of the linker with the dye along the inner surface of the ion channel.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

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Section: Synthesis Of the Probesmentioning

confidence: 98%

Synthesis of Small‐Molecule Fluorescent Probes for the In Vitro Imaging of Calcium‐Activated Potassium Channel K_Ca3.1

et al. 2020

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“…EF-hands are found within the cytoplasmic regions of these channel proteins and alter channel function upon localized calcium accumulation. In contrast, the small conductance Ca 2+ activated K+ channel, SK (Lee and MacKinnon, 2018) and the subfamily of KCNQ voltage-gated K + channels, Kv7.4 (Chang et al, 2018) forms a complex with Ca 2+ binding protein, CaM, which regulates Ca 2+ dependent activation.…”

Section: Discussionmentioning

confidence: 99%

EFCAB9 is a pH-Dependent Ca²⁺Sensor that Regulates CatSper Channel Activity and Sperm Motility

Hwang

Mannowetz²,

Zhang

et al. 2018

Preprint

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16Varying pH of luminal fluid along the female reproductive tract is a physiological cue that 17 modulates sperm motility. CatSper is a sperm-specific, pH-sensitive calcium channel essential 18 for hyperactivated motility and male fertility. Multi-subunit CatSper channel complexes organize 19 linear Ca 2+ signaling nanodomains along the sperm tail. Here, we identify EF-hand calcium-20 binding domain-containing protein 9 (EFCAB9) as a dual function, cytoplasmic machine 21 modulating the channel activity and the domain organization of CatSper. Knockout mice studies 22 demonstrate that EFCAB9, in complex with the CatSper subunit, CATSPER, is essential for pH-23 dependent and Ca 2+ sensitive activation of the CatSper channel. In the absence of EFCAB9, 24 sperm motility and fertility is compromised and the linear arrangement of the Ca 2+ signaling 25 domains is disrupted. EFCAB9 interacts directly with CATSPER in a Ca 2+ dependent manner 26 and dissociates at elevated pH. These observations suggest that EFCAB9 is a long-sought, 27 intracellular, pH-dependent Ca 2+ sensor that triggers changes in sperm motility. 28 29 Keywords (up to 10 keywords) 30 Sperm motility, male fertility, Ca 2+ channel, CatSper, pH sensing, Ca 2+ sensor, Ca 2+ signal 31 transduction 32 33 Highlights 34  Efcab9 encodes an evolutionarily conserved, sperm-specific EF-hand domain protein 35  Efcab9-deficient mice have sperm motility defects and reduced male fertility 36  EFCAB9 is a pH-tuned Ca 2+ sensor for flagellar CatSper Ca 2+ channel 37  EFCAB9 is a dual function machine in gatekeeping and domain organization of CatSper 38 39 82super-resolution imaging levels. We find that EFCAB9 is a pH-dependent Ca 2+ sensor for the 83 CatSper channel. EFCAB9-CatSper forms a binary complex and interacts with the pore as a 84 gatekeeper, activating CatSper channel responding to capacitation-associated intracellular 85 changes. EFCAB9 interaction with CatSper requires its Ca 2+ binding to EF-hands, which are 86 responsive to pH changes in a physiologically relevant range. Loss of EFCAB9-CatSper largely 87 Hwang et al.,2018 3 eliminates the pH-dependent and Ca 2+ sensitive activation of CatSper and alters newly resolved 88 CatSper Ca 2+ signaling domains. In contrast to the recent addition of CatSper to mammals, the 89 EFCAB9 and other transmembrane CatSper subunits are evolutionarily conserved from 90 flagellated single cell eukaryotes. These findings provide insight into evolutionarily conserved 91 gating mechanisms for CatSper channels and reveal an adaptation for Ca 2+ signaling in flagella. 92 93 RESULTS 94 95 97 Sperm hyperactivated motility requires pH-dependent activation of the CatSper flagellar Ca 2+ 98 channel. The CatSper channel forms a multi-protein complex composed of at least nine subunits 99 (CatSper1-4, , , , , and ) (Chung et al., 2017). Here we examine a new member of the 100 complex and determine its functional significance. 101 102Our previous studies showed that other CatSper subunits were not detected in CatSper1-a...

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“…The channel lacks a voltage sensor domain and is therefore not voltage dependent, an important feature that allows the channel to evoke a robust hyperpolarisation response when the cell is activated, during which time the membrane potential approaches −80 mV, the equilibrium potential of K + (Grgic, Kaistha, Hoyer, & Kohler, ). Recently, Lee and Mackinnon et al () have examined the structure of K Ca 3.1 in closed and activated states via cryogenic electron microscopy, providing significant insight into the gating mechanisms of K Ca 3.1 (Lee & Mackinnon, ).…”

Section: Introductionmentioning

confidence: 99%

“…Cryogenic electron microscopy has shown that in the absence of Ca 2+ , the K Ca 3.1 channel pore remains closed by maintaining the resting state. Specifically, CaM stays associated with the channel through its C‐lobe, while the CaM N‐lobe maintains only weak interactions with the channel and is conformationally flexible (Lee & MacKinnon, ). When there is an increase in Ca 2+ concentration, the CaM N‐lobe binds to Ca 2+ triggering a conformational change, increasing the affinity of the N‐lobe for the S 45 A helix.…”

Section: Introductionmentioning

confidence: 99%

“…When there is an increase in Ca 2+ concentration, the CaM N‐lobe binds to Ca 2+ triggering a conformational change, increasing the affinity of the N‐lobe for the S 45 A helix. It is this interaction of S 45 A and the N‐lobe that causes S 45 B to move away from the pore axis, rearranging the S6 helices and permitting the pore to open (Lee & MacKinnon, ).…”

Section: Introductionmentioning

confidence: 99%

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Ca²⁺ signalling in fibroblasts and the therapeutic potential of K_Ca3.1 channel blockers in fibrotic diseases

Roach

Bradding

2020

British J Pharmacology

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The role of Ca2+ signalling in fibroblasts is of great interest in fibrosis‐related diseases. Intracellular free Ca2+ ([Ca2+]i) is a ubiquitous secondary messenger, regulating a number of cellular functions such as secretion, metabolism, differentiation, proliferation and contraction. The intermediate conductance Ca2+‐activated K+ channel KCa3.1 is pivotal in Ca2+ signalling and plays a central role in fibroblast processes including cell activation, migration and proliferation through the regulation of cell membrane potential. Evidence from a number of approaches demonstrates that KCa3.1 plays an important role in the development of many fibrotic diseases, including idiopathic pulmonary, renal tubulointerstitial fibrosis and cardiovascular disease. The KCa3.1 selective blocker senicapoc was well tolerated in clinical trials for sickle cell disease, raising the possibility of rapid translation to the clinic for people suffering from pathological fibrosis. This review after analysing all the data, concludes that targeting KCa3.1 should be a high priority for human fibrotic disease.

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Activation mechanism of a human SK-calmodulin channel complex elucidated by cryo-EM structures

Cited by 144 publications

References 78 publications

Synthesis of Small‐Molecule Fluorescent Probes for the In Vitro Imaging of Calcium‐Activated Potassium Channel K_Ca3.1

Synthesis of Small‐Molecule Fluorescent Probes for the In Vitro Imaging of Calcium‐Activated Potassium Channel K_Ca3.1

EFCAB9 is a pH-Dependent Ca²⁺Sensor that Regulates CatSper Channel Activity and Sperm Motility

Ca²⁺ signalling in fibroblasts and the therapeutic potential of K_Ca3.1 channel blockers in fibrotic diseases

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Activation mechanism of a human SK-calmodulin channel complex elucidated by cryo-EM structures

Cited by 144 publications

References 78 publications

Synthesis of Small‐Molecule Fluorescent Probes for the In Vitro Imaging of Calcium‐Activated Potassium Channel KCa3.1

Synthesis of Small‐Molecule Fluorescent Probes for the In Vitro Imaging of Calcium‐Activated Potassium Channel KCa3.1

EFCAB9 is a pH-Dependent Ca2+Sensor that Regulates CatSper Channel Activity and Sperm Motility

Ca2+ signalling in fibroblasts and the therapeutic potential of KCa3.1 channel blockers in fibrotic diseases

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Product

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Synthesis of Small‐Molecule Fluorescent Probes for the In Vitro Imaging of Calcium‐Activated Potassium Channel K_Ca3.1

Synthesis of Small‐Molecule Fluorescent Probes for the In Vitro Imaging of Calcium‐Activated Potassium Channel K_Ca3.1

EFCAB9 is a pH-Dependent Ca²⁺Sensor that Regulates CatSper Channel Activity and Sperm Motility

Ca²⁺ signalling in fibroblasts and the therapeutic potential of K_Ca3.1 channel blockers in fibrotic diseases