Two rings of negative charges in the cytosolic vestibule of type-1 ryanodine receptor modulate ion fluxes

¹ University of North Carolina
² Rush University Medical Center

^* To whom correspondence should be addressed. E-mail: meissner{at}med.unc.edu.

Submitted on August 11, 2005
Revised on September 20, 2005
Accepted on 4 October 2005

	Abstract

The tetrameric ryanodine receptor calcium release channels (RyRs) are cation-selective channels that have pore architecture similar to that of K+ channels. We recently identified, in close proximity to the selectivity filter motif GGGIG, a conserved lumenal DE motif that has a critical role in RyR ion permeation and selectivity. Here, we substituted three aspartate residues (D4938, D4945, D4953) with asparagine and four glutamate residues (E4942, E4948, E4952, E4955) with glutamine hypothesized to line the cytosolic vestibule of the skeletal muscle RyR (RyR1). Mutant single channel properties were determined using the planar lipid bilayer method. Two mutants (D4938N, D4945N) showed a reduced K+ ion conductance, with D4938N also exhibiting a reduced selectivity for Ca2+ compared to K+. The cytosolic location of D4938 and D4945 was confirmed using the polycation neomycin. Both D4938N and D4945N exhibited attenuated block by neomycin to a greater extent from the cytosolic than lumenal side. By comparison, charge neutralization of lumenal loop residues (D4899Q, E4900N) eliminated the block from the lumenal but not cytosolic side. The results suggest that, in addition to negatively charged residues on the lumenal side, rings of four negative charges formed by D4938 and D4945 in the cytosolic vestibule, determine RyR ion fluxes.

Key Words: Ca2+ release channel, ion permeation, ion selectivity, ryanodine receptor, skeletal muscle

This article has been cited by other articles:

				D. Gillespie and D. Boda The Anomalous Mole Fraction Effect in Calcium Channels: A Measure of Preferential Selectivity Biophys. J., September 15, 2008; 95(6): 2658 - 2672. [Abstract] [Full Text] [PDF]

				J. Qin, G. Valle, A. Nani, A. Nori, N. Rizzi, S. G. Priori, P. Volpe, and M. Fill Luminal Ca2+ Regulation of Single Cardiac Ryanodine Receptors: Insights Provided by Calsequestrin and its Mutants J. Gen. Physiol., March 31, 2008; 131(4): 325 - 334. [Abstract] [Full Text] [PDF]

				L. Xu, Y. Wang, N. Yamaguchi, D. A. Pasek, and G. Meissner Single Channel Properties of Heterotetrameric Mutant RyR1 Ion Channels Linked to Core Myopathies J. Biol. Chem., March 7, 2008; 283(10): 6321 - 6329. [Abstract] [Full Text] [PDF]

				D. Gillespie Energetics of Divalent Selectivity in a Calcium Channel: The Ryanodine Receptor Case Study Biophys. J., February 15, 2008; 94(4): 1169 - 1184. [Abstract] [Full Text] [PDF]

				O. Dellis, A. M. Rossi, S. G. Dedos, and C. W. Taylor Counting Functional Inositol 1,4,5-Trisphosphate Receptors into the Plasma Membrane J. Biol. Chem., January 11, 2008; 283(2): 751 - 755. [Abstract] [Full Text] [PDF]

				D. Boda, W. Nonner, M. Valisko, D. Henderson, B. Eisenberg, and D. Gillespie Steric Selectivity in Na Channels Arising from Protein Polarization and Mobile Side Chains Biophys. J., September 15, 2007; 93(6): 1960 - 1980. [Abstract] [Full Text] [PDF]

				J. K. Foskett, C. White, K.-H. Cheung, and D.-O. D. Mak Inositol Trisphosphate Receptor Ca2+ Release Channels Physiol Rev, April 1, 2007; 87(2): 593 - 658. [Abstract] [Full Text] [PDF]