PROBING THE STRUCTURE OF THE CONDUCTION PATHWAY OF THE SHEEP CARDIAC SARCOPLASMIC-RETICULUM CALCIUM-RELEASE CHANNEL WITH PERMEANT AND IMPERMEANT ORGANIC CATIONS

The sarcoplasmic reticulum Ca2+-release channel plays a central role in cardiac muscle function by providing a ligand-regulated pathway for the release of sequestered Ca2+ to initiate contraction following cell excitation. The efficiency of the channel as a Ca2+-release pathway will be influenced by both gating and conductance properties of the system. In the past we have investigated conduction and discrimination of inorganic mono- and divalent cations with the aim of describing the mechanisms governing ion handling in the channel (Tinker, A., A. R. G. Lindsay, and A. J. Williams. 1992. Journal of General Physiology. 100:495-517.). In the present study, we have used permeant and impermeant organic cations to provide additional information on structural features of the conduction pathway. The use of permeant organic cations in biological channels to explore structural motifs underlying selectivity has been an important tool for the electrophysiologist. We have examined the conduction properties of a series of monovalent organic cations of varying size in the purified sheep cardiac sarcoplasmic reticulum Ca2+-release channel. Relative permeability, determined from the reversal potential measured under bi-ionic conditions with 210-mM test cation at the cytoplasmic face of the channel and 210 mM K+ at the luminal, was related inversely to the minimum circular cation radius. The reversal potential was concentration-independent. The excluded area hypothesis, with and without a term for solute-wall friction, described the data well and gave a lower estimate for minimum pore radius of 3.3-3.5 angstrom. Blocking studies with the impermeant charged derivative of triethylamine reveal that this narrowing occurs over the first 10-20% of the voltage drop when crossing from the lumen of the SR to the cytoplasm. Single-channel conductances were measured in symmetrical 210 mM salt. Factors other than relative permeability determine conductance as ions with similar relative permeability can have widely varying single-channel conductance. Permeant ions, such as the charged derivatives of trimethylamine and diethylmethylamine, can also inhibit K+ current. The reduction in relative conductance with increasing concentrations of these two ions at a holding potential of 60 mV was described by a rectangular hyperbola and revealed higher affinity binding for diethylmethylamine as compared to trimethylamine. It was possible to describe the complex permeation properties of these two ions using a single-ion four barrier, three binding site Eyring rate theory model. In conclusion, these studies reveal that the cardiac Ca2+-release channel has a selectivity filter of approximately 3.5-angstrom radius located at the luminal face of the protein. Transport rates for organic cations are determined by sieving according to size at the selectivity filter, with specific chemical factors playing only a small role, and a hydrophobic binding site located just after this as cations pass from the lumen of the sarcoplasmic reticulum to the cytosol.