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 i n 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 w ill be influenced by both gating and conductance properties of the sys tem. In the past we have investigated conduction and discrimination of inorganic mono- and divalent cations with the aim of describing the m echanisms 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 featur es of the conduction pathway. The use of permeant organic cations in b iological channels to explore structural motifs underlying selectivity has been an important tool for the electrophysiologist. We have exami ned the conduction properties of a series of monovalent organic cation s of varying size in the purified sheep cardiac sarcoplasmic reticulum Ca2+-release channel. Relative permeability, determined from the reve rsal potential measured under bi-ionic conditions with 210-mM test cat ion at the cytoplasmic face of the channel and 210 mM K+ at the lumina l, was related inversely to the minimum circular cation radius. The re versal potential was concentration-independent. The excluded area hypo thesis, with and without a term for solute-wall friction, described th e data well and gave a lower estimate for minimum pore radius of 3.3-3 .5 angstrom. Blocking studies with the impermeant charged derivative o f 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 cy toplasm. Single-channel conductances were measured in symmetrical 210 mM salt. Factors other than relative permeability determine conductanc e as ions with similar relative permeability can have widely varying s ingle-channel conductance. Permeant ions, such as the charged derivati ves of trimethylamine and diethylmethylamine, can also inhibit K+ curr ent. The reduction in relative conductance with increasing concentrati ons of these two ions at a holding potential of 60 mV was described by a rectangular hyperbola and revealed higher affinity binding for diet hylmethylamine as compared to trimethylamine. It was possible to descr ibe the complex permeation properties of these two ions using a single -ion four barrier, three binding site Eyring rate theory model. In con clusion, these studies reveal that the cardiac Ca2+-release channel ha s 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 hydro phobic binding site located just after this as cations pass from the l umen of the sarcoplasmic reticulum to the cytosol.