C1 - Calcium release channels

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Calcium release channels experiments

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Sitsapesan & Colleagues aim

Sitsapesan & Colleagues methods

Sitsapesan & Colleagues findings

Kermode aim

Kermode methods

Kermode findings

Chen aim

Chen methods

Chen findings

Yan aim

Yan methods

Yan findings

Gillespie aim

Gillespie methods

Gillespie findings

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site directed mutagenesis @ C-terminus -ive residues. in vivo het knock-in of mutation found to abolish luminal sensing.

Determine nature/location of luminal Ca-sensor preserved on RyR/IP3R

ATP -> activates RyR through Ca-dependent & Ca-independent mechs. ADP partial agonist.

describe regulation of native RyR by ATP/ADP

A countercurrent is necessary to stop changes in Vm that would be inhibitory to Ca fluxes associated with sparks. RyR is known to be poorly selective to Ca, and a robust model of ion permeation through a single RyR (described by Gillespie) predicts a significant K+ counterflux under physiological conditions

effects of FKBP12 & FKBP12.6 on RyR determined, non-homologous residues assessed, FKBP12 mutant generated that acts like FKBP12.6

Proposed the nature of the countercurrent for RyR

ATP --> activates RyR (maximal 2mM, bell shaped) w/\Ca, gating kinetics differ w\. ADP + ATP limits efficacy of ATP in activating.

cryo-EM was used to describe the channel in a closed state

RyR mediates its own countercurrent by mediating K+ influx to the SR

Determine near-atomic structure of RyR1

described a closed state in which the four S6 segments constrict at the inner activation gate. ion conducting pathway formed by S6 segments & selectivity filter, spans ~90A. adjacent to selectivity filter = -vely charged residues thought to attract cations.

FKBP12-> activates RyR2, inhibits RyR1. FKBP12.6-> activates RyR1, partial agonist @ RyR2. Three residues found to determine these differences.

describe regulation of RyR by FKBPs

E4872Q mutation abolishes luminal Ca sensitivity in vitro & protects against SOICR in vivo.


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