Lanthanides Report Calcium Sensor in the Vestibule of Ryanodine Receptor.

Abstract:

:Ca2+ regulates ryanodine receptor's (RyR) activity through an activating and an inhibiting Ca2+-binding site located on the cytoplasmic side of the RyR channel. Their altered sensitivity plays an important role in the pathology of malignant hyperthermia and heart failure. We used lanthanide ions (Ln3+) as probes to investigate the Ca2+ sensors of RyR, because they specifically bind to Ca2+-binding proteins and they are impermeable to the channel. Eu3+'s and Sm3+'s action was tested on single RyR1 channels reconstituted into planar lipid bilayers. When the activating binding site was saturated by 50 μM Ca2+, Ln3+ potently inhibited RyR's open probability (Kd Eu3+ = 167 ± 5 nM and Kd Sm3+ = 63 ± 3 nM), but in nominally 0 [Ca2+], low [Eu3+] activated the channel. These results suggest that Ln3+ acts as an agonist of both Ca2+-binding sites. More importantly, the voltage-dependent characteristics of Ln3+'s action led to the conclusion that the activating Ca2+ binding site is located within the electrical field of the channel (in the vestibule). This idea was tested by applying the pore blocker toxin maurocalcine on the cytoplasmic side of RyR. These experiments showed that RyR lost reactivity to changing cytosolic [Ca2+] from 50 μM to 100 nM when the toxin occupied the vestibule. These results suggest that maurocalcine mechanically prevented Ca2+ from dissociating from its binding site and support our vestibular Ca2+ sensor-model further.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Sárközi S,Komáromi I,Jóna I,Almássy J

doi

10.1016/j.bpj.2017.03.023

subject

Has Abstract

pub_date

2017-05-23 00:00:00

pages

2127-2137

issue

10

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(17)30342-9

journal_volume

112

pub_type

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