Physical dimensions of the pores of VDACL, VSOR and CFTR chloride channels as putative ATP-channels.

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Maxi-anion channel, volume-sensitive outwardly rectifying (VSOR) anion channel and cystic fibrosis transmembrane conductance regulator (CFTR) channel have been suggested to mediate an electrogenic transport of ATP by different groups. This function requires a pore wide enough for passing a bulky ATP molecule. The purpose of the present project was to evaluate the physical dimensions of the pores of the three putative ATP-channels. (1) Single maxi-anion channels were recorded from membrane patches excised from mouse mammary C127 cells. We first analyzed the permeability of the channel to a series of organic anions of different size. We found a linear relationship between relative permeability of organic anions of different size and their relative ionic mobility (measured as the ratio of ionic conductance) with a slope close to 1, suggesting that organic anions tested with radii up to 0.49 nm (lactobionate) move inside the channel by free diffusion. In the second approach, we, for the fi … More rst time, succeeded in pore sizing by the nonelectrolyte exclusion method in single-channel patch-clamp experiments. The cut-off radii of PEG molecules that could access the channel from intracellular (1.16 nm) and extracellular (1.42 nm) sides indicated an asymmetry of the two entrances to the channel pore. Measurements by symmetrical two-sided application of PEG molecules yielded an average functional pore radius of 〜1.3 nm. (2) Single VSOR channels were recorded from the cell-attached patches of human epithelial Intestine 407 cells pre-swollen in hypotonic Hi-K+ solutions. PEG 200-300 (Rh=0.27-0.53 nm) effectively suppressed the single-channel currents, whereas PEG 400-4000 (Rh=0.62-1.91 nm) had little or no effect. The cut-off radius of the VSOR channel pore was assessed to be 0.63 nm. (3) Single CFTR channels were recorded from cell-attached and excised membrane patches from HEK293T cells transiently transfected with CFTR gene using a bi-cistronic vector with GFP gene as a reporter. The effect of polyethylene glycols on the single-channel CFTR currents was different depending on whether they were added from extracellular or intracellular side. The cut-off radius of PEG molecules that could access the channel from the intracellular side (〜1.0 nm) was larger than that from the extracellular side (〜0.6 nm) indicating an asymmetry of the two channel entrances.The radius of ATP4- and MgATP2- (about 0.6-0.7 nm) is close to the limiting size of the VSOR channel pore, and narrowest part of CFTR channel. The size of maxi-anion channel (〜1.3 nm) is largest among these three channels; it is best suited to its function as an ATP channel and, where present, serves as the preferred pathway for release of ATP4- and/or MgATP2-. Less