Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC262
Location of the tetraalkyl ammonium (TAA) blocking site in the mouse cardiac ryanodine receptor (mRyR2)
S. A. Mason1, D. West2, C. Viero1, S. Chen3, A. Williams1
1. Cardiology, Wales Heart Research Institute, Cardiff, South Glamorgan, United Kingdom. 2. Cardiac Medicine, National Heart and Lung Institute, London, United Kingdom. 3. Physiology and Pharmacology, Libin Cardiovascular Institute, Calgary, Alberta, Canada.
In the absence of structural details for the RyR2 pore we have developed an analogy model using KcsA as a template (1). Here we test the model’s prediction that the cytosolic cavity of RyR2 is lined by hydrophobic residues of the 10th trans-membrane domain (TM10) by monitoring block by large TAAs. We hypothesise that increases in hydrophobicity of TAAs will result in higher affinity block due to decreased rates of dissociation (koff). An ensuing prediction is that TM10 mutants with decreased hydrophobicity (I4857A, I4861A, I4862A, L4865A, I4867A, F4870A and L4873A) will have reduced affinities for TAAs owing to increases in koff. mRyR2 was expressed and purified as described previously (2) and incorporated into phosphatidylethanolamine bilayers for voltage clamp experiments. Single channel recordings were made in symmetrical 610 mM KCl, 20 mM HEPES, 10 μM Ca2+ at pH 7.2 and 22°C. To characterise block, channels were activated to open probabilities approaching 1.0 either by 20 µM EMD41000 (EMD) or by modification with 1 µM ryanodine. TAAs were added to both sides of mRyR2 at 10-200 µM. Analyses of block and current amplitudes were described previously (3). Data are expressed as Mean±SEM. Variations in blocking parameters of TAAs (tetrabutyl (TBA), tetrapentyl (TPeA) and tetrahexyl (THexA)) were determined in EMD-activated wild type (WT) mRyR2. TAAs induce subconductance block at positive potentials and residual current diminishes as TAA size increases (TBA: 18.51±0.14; TPeA: 10.89±0.22; THexA: 3.47± 0.65% of full amplitude, n=4-5). mRyR2 closes whilst blocked and the probability of closure is unaffected by block. Analysis of blocking events indicates that increases in hydrophobicity have no effect on rates of TAA association but significantly decrease koff (p<0.05) (TBA: 412.2±24.5; TPeA: 18.6±1.1; THexA: 11.1±1.6 s-1, n=4-5). Single channel K+ conductance was not affected by substitution of TM10 hydrophobic residues with alanine however the ability of EMD to fully activate the channel was compromised in some cases. The influence of lowering residue hydrophobicity was characterised by monitoring block by TPeA in EMD-activated and/or ryanodine-modified channels. In channels that could be activated by EMD residue substitution resulted in a lowering of TPeA affinity as the result of a significant increase in koff (WT: 18.5±1.1; I4861A: 26.4±2.0; I4862A: 45.6±5.0; L4865A 21.2±0.9 s-1, p<0.05, n =5-6). Following ryanodine modification all mutants exhibited a decreased affinity for TPeA and the majority exhibited significant increases in koff (WT: 35.6±0.9; I4861A: 77.4±6.1; I4862A: 77.6±1.4; L4865A: 52.3±3.4; I4867A: 42.0±1.8; F4870A: 62.4±9.1 and L4873A: 93.5±5.6 s-1, p<0.05, n=5-8). Our data support the proposal that blockade by TAAs is stabilised by interactions with hydrophobic residues of TM10 lining the cytosolic cavity of the RyR2 pore.
Where applicable, experiments conform with Society ethical requirements