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Changes in negative charge at the luminal mouth of the pore alter ion handling and gating in the cardiac ryanodine-receptor

Mead-Savery, Fiona C., Wang, Ruiwu, Tanna-Topan, Bhavna, Chen, S. R. Wayne, Welch, William and Williams, Alan John 2009. Changes in negative charge at the luminal mouth of the pore alter ion handling and gating in the cardiac ryanodine-receptor. Biophysical journal 96 (4) , pp. 1374-1387. 10.1016/j.bpj.2008.10.054

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We have tested the hypothesis that a high density of negative charge at the luminal mouth of the RyR2 pore plays a pivotal role in the high cation conductance and limited selectivity observed in this channel by introducing into each monomer a double point mutation to neutralize acidic residues in this region of the mouse RyR2 channel. The resultant channel, ED4832AA, is capable of functioning as a calcium-release channel in situ. Consistent with our hypothesis, the ED4832AA mutation altered the ion handling characteristics of single RyR2 channels. The mutant channel retains the ability to discriminate between cations and anions but cation conductance is altered significantly. Unitary K+ conductance is reduced at low levels of activity but increases dramatically as activity is raised and shows little sign of saturation. ED4832AA no longer discriminates between divalent and monovalent cations. In addition, the gating characteristics of single RyR2 channels are altered markedly by residue neutralization. Open probability in the ED4832AA channel is substantially higher than that of the wild-type channel. Moreover, at holding potentials in excess of 50 mV several subconductance states become apparent in ED4832AA and are more prevalent at very high holding potentials. These observations are discussed within the structural framework provided by a previously developed model of the RyR2 pore. Our data indicates that neutralization of acidic residues in the luminal mouth of the pore produces wide-ranging changes in the electric field in the pore, the interaction energies of permeant ions in the pore and the stability of the selectivity filter region of the pore, which together contribute to the observed changes ion handling and gating.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Subjects: R Medicine > R Medicine (General)
Publisher: Biophysical Society
ISSN: 0006-3495
Last Modified: 12 Jun 2019 02:29

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