Methanethiosulfonate Ethylammonium Block of Amine Currents through the Ryanodine Receptor Reveals Single Pore Architecture

Abstract

The homotetrameric structure of the ryanodine-sensitive intracellular calcium (Ca 2؉ ) 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.

Organic Cation Permeation through the Channel Formed by Polycystin-2

“…2A). This observation is similar to that seen when organic amines of varying sizes conduct through the intracellular calcium channel, the RyR (11,13,19).…”

“…To investigate this difference, we examined the permeation properties of PC2. In previous reports, organic cations with known ionic diameters were employed to determine the pore diameters of ion channels such as the ryanodine receptor (RyR), potassium channel KcsA, and the vanilloid receptor (TRPV6) (11)(12)(13)(14). These previous reports revealed that the RyR has one pore of a minimum diameter of ϳ7 Å (13).…”

Regulation of ryanodine receptor-dependent calcium signaling by polycystin-2

“…Mouse hearts were isolated from adult mice as described (42). For immunoblotting experiments, mouse heart lysates were solubilized with 1% CHAPS for 1 h under constant agitation.…”

“…The Ca 2ϩ concentration was calculated and presented as Ϫlog[Ca 2ϩ ] (45). Channel data were acquired as described (42). Open probability was determined by using current recordings from at least 2 s of continuously recorded data.…”

“…SR vesicles were isolated from canine heart and incorporated into planar lipid bilayer as described (29,42). Single-channel activity of RyR2 was measured in the presence or absence of GST-CPC2 or GST-NPC2.…”