M2 MUTATIONS, WHICH SWITCH THE ION SELECTIVITY OF GLYCINE RECEPTOR CHANNELS AND ACT VIA BOTH ELECTROSTATIC EFFECTS AND CHANGES IN MINIMUM PORE DIAMETER, AND MATHEMATICAL MODELLING
A. Keramidas1, A.J. Moorhouse1, M. O'Mara3, K.D. Pierce2, P.R. Schofield2, S.H. Chung3,
P.H. Barry1, 1 Dept of Physiology & Pharmacology, University of New South Wales, Sydney 2052, 2 The Garvan Institute of Medical Research, Darlinghurst, Sydney 2010, 3 Dept of Theoretical Physics, Australian National University, ACT 0200.
Patch clamp studies of recombinant glycine receptor (GlyR) channels expressed in HEK293 cells have previously enabled us to show that a series of (single, double and triple) point mutations in the M2 region of the glycine receptor were able to each switch the GlyR selectivity from being anion- to cation-selective 1,2,3. Dilution potential measurements were used to determine the permeability ratio (PCl/PNa) and bi-ionic potential measurements (with organic cations) to determine minimum pore diameters (MPD) of the mutant GlyR channels. A single amino acid substitution that introduces a negatively charged ring of glutamate residues (A-1'E) adjacent to ring of arginines (R0') was able to switch the ion selectivity with PCl/PNa changing from ∼25-28 (WT, Wild Type) to ∼0.342 with pore diameter changing from 5.3 Å (WT)4 to 6.5 Å (A-1'E GlyR)2. In the selectivity double mutation (SDM; P-2'Δ & A-1'E), the deletion of the adjacent proline residues decreased PCl/PNa to ∼0.13 and produced an MPD of 9.7 Å A triple mutation, which neutralizes the external ring of charge (SDMR19'A) had a PCl/PNa to ∼0.23 and an MPD of 8.2 Å whereas the (SDM+R19'E) had a PCl/PNa ∼0.19 and an MPD of 8.3 Å These results all indicate that the determinants of ion charge selectivity include electrostatic effects and changes in minimum pore diameter at the selectivity filter of the GlyR channel. We have now started to mathematically model these ion permeation studies using Brownian dynamics simulations5.
(1) Keramidas A, Moorhouse AJ, French CR, Schofield PR, Barry PH. Biophysical Journal. 2000; 78:247-259.
(2) Keramidas A, Moorhouse AJ, Pierce, KD, Schofield PR, Barry PH. Journal of General Physiology. 2002; 119:393-410.
(3) Moorhouse AJ, Keramidas A, Zaykin A, Schofield PR, Barry PH. Journal of General Physiology. 2002; 119:411-425.
(4) Rundstrom N, Schmieden V, Betz H, Bormann J, Langosch D. Proceedings of the National Academy of Science, USA. 1994; 91:8950-8954.
(5) Chung SH, Kuyucak S. European Biophysical Journal. 2002; 31:283-293.
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