Methanethiosulfonate ethylammonium block of amine currents through the ryanodine receptor reveals single pore architecture.
The homotetrameric structure of the ryanodine-sensitive intracellular calcium (Ca2+) release channel (ryanodine receptor (RyR)) suggests that the four RyR subunits either combine to form a single pore or that each RyR subunit is an independently conducting pathway. Previously we showed that methanethiosulfonate ethylammonium (MTSEA+) covalently modifies the RyR to reduce current amplitudes in a time-dependent and stepwise manner. To ascertain the number of functionally conducting pores in the RyR, two approaches were combined: modification of the receptor by MTSEA+ and the use of different sized current carriers. Previous reports (Tinker, A., and Williams, A. J. (1993) J. Gen. Physiol. 102, 1107-1129) have shown that the organic cations methylamine, dimethylamine, ethylamine, and trimethylamine are permeant through the RyR but with reduced current amplitude depending upon the diameter of the respective amine. Experiments using the thiol reagent MTSEA+ to modify the channel protein showed that the current amplitudes decrease in steps leading to complete block of the channel when cesium (Cs+) is the current carrier. MTSEA+ modification decreased the number of channel substates as the diameter of the current carrier increased. Comparison of the degree of inhibition of MTSEA+-modified currents allows for differentiation between the two models for channel architecture. These results demonstrate that the conduction pathway for the RyR is comprised of a single central pore.
Anyatonwu
Georgia I
GI
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8356, USA.
Buck
Edmond D
ED
Ehrlich
Barbara E
BE
D000588:Amines / Q000737:chemistry*; D000818:Animals; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D002586:Cesium / Q000494:pharmacology; D004396:Coloring Agents / Q000494:pharmacology; D004285:Dogs; D004594:Electrophysiology; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D007477:Ions; D008051:Lipid Bilayers / Q000378:metabolism; D008744:Methylamines / Q000737:chemistry; D008954:Models, Biological; D009206:Myocardium / Q000378:metabolism; D012430:Ruthenium Red / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013997:Time Factors
D000588:Amines; D002412:Cations; D004396:Coloring Agents; D007477:Ions; D008051:Lipid Bilayers; D008744:Methylamines; D019837:Ryanodine Receptor Calcium Release Channel; D013438:Sulfhydryl Compounds; C093171:methanethiosulfonate ethylammonium; D012430:Ruthenium Red; D002586:Cesium; D005020:Ethyl Methanesulfonate; C027451:methylamine