HOW DOE RYANODINE MODIFY ION HANDLING IN THE SHEEP CARDIAC SARCOPLASMIC-RETICULUM CA2-RELEASE CHANNEL()

Under appropriate conditions, the interaction of the plant alkaloid ry anodine with a single cardiac sarcoplasmic reticulum Ca2+-release chan nel results in a profound modification of both channel gating and cond uction. On modification, the channel undergoes a dramatic increase in open probability and a change in single-channel conductance. In this p aper we aim to provide a mechanistic framework for the interpretation of the altered conductance seen after ryanodine binding to the channel protein. To do this we have characterized single-channel conductance with representative members of three classes of permeant cation; group 1a monovalent cations, alkaline earth divalent cations, and organic m onovalent cations. We have quantified the change in single-channel con ductance induced by ryanodine and have expressed this as a fraction of conductance in the absence of ryanodine. Fractional conductance seen in symmetrical 210 mM solutions is not fixed but varies with the natur e of the permeant cation. The group 1a monovalent cations (K+, Na+, Cs +, Li+) have values of fractional conductance in a narrow range (0.60- 0.66). With divalent cations fractional conductance is considerably lo wer (Ba2+, 0.22 and Sr2+, 0.28), whereas values of fractional conducta nce vary considerably with the organic monovalent cations (ammonia 0.6 6, ethylamine 0.76, propanolamine 0.65, diethanolamine 0.92, diethylam ine 1.2). To establish the mechanisms governing these differences, we have monitored the affinity of the conduction pathway for, and the rel ative permeability of, representative cations in the ryanodine-modifie d channel. These parameters have been compared with those obtained in previous studies from this laboratory using the channel in the absence of ryanodine and have been modeled by modifying our existing single-i on, four-barrier three-well rate theory model of conduction in the unm odified channel. Our findings indicate that the high affinity, essenti ally irreversible, interaction of ryanodine with the cardiac sarcoplas mic reticulum CA(2+)-release channel produces a conformational alterat ion of the protein which results in modified ion handling. We suggest that, on modification, the affinity of the channel for the group 1a mo novalent cations is increased while the relative permeability of this class of cations remains essentially unaltered. The affinity of the co nduction pathway for the alkaline earth divalent cations is also incre ased, however the relative permeability of this class of cations is re duced compared to the unmodified channel. The influence of modificatio n on the handling by the channel of the organic monovalent cations is determined by both the size and the nature of the cation. Small cation s such as ammonia respond to ryanodine-induced alterations of the cond uction pathway in much the same way as the group 1a monovalents. Large r organic cations such as diethanolamine and diethylamine have an incr eased relative permeability following ryanodine modification. Conducta nce of these cations is also influenced by the relative importance of their interactions with both hydrophobic and hydrophilic binding sites of the conduction pathway. These altered parameters of ion handling h ave been incorporated into an Eyring rate theory model and provide an adequate description of the observed variation in fractional conductan ce seen on modification of the channel by ryanodine in the presence of the different classes of permeant cation.