It has been demonstrated earlier that the neural network based program AlphaFold2 can be used to dock proteins given the two sequences separated by a gap as the input. The protocol presented here combines AlphaFold2 with the physics based docking program ClusPro. The monomers of the model generated by AlphaFold2 are separated, re-docked using ClusPro, and the resulting 10 models are refined by AlphaFold2. Finally, the five original AlphaFold2 models are added to the 10 AlphaFold2 refined ClusPro models, and the 15 models are ranked by their predicted aligned error (PAE) values obtained by AlphaFold2. The protocol is applied to two benchmark sets of complexes, the first based on the established protein-protein docking benchmark, and the second consisting of only structures released after May 2018, the cut-off date for training AlphaFold2. It is shown that the quality of the initial AlphaFold2 models improves with each additional step of the protocol. In particular, adding the AlphaFold2 refined ClusPro models to the AlphaFold2 models increases the success rate by 23% in the top 5 predictions, whereas considering the 10 models obtained by the combined protocol increases the success rate to close to 40%. The improvement is similar for the second benchmark that includes only complexes distinct from the proteins used for training the neural network.

When human erythrocytes are suspended in low-Cl- media (with sucrose replacing Cl-), there is a large increase in both the net efflux and permeability of K+. A substantial portion (greater than 70% with Cl- less than 12.5 mM) of this K+ efflux is inhibited by the anion exchange inhibitor DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). This inhibition cannot be explained as an effect of DIDS on net Cl- permeability (Pcl) and membrane potential, but rather represents a direct effect on the K+ permeability. When cells are reacted with DIDS for different times, the inhibition of K+ efflux parallels that of Cl- exchange, which strongly indicates that the band 3 anion exchange protein (capnophorin) mediates the net K+ flux. Since a noncompetitive inhibitor of anion exchange, niflumic acid, has no effect on net K+ efflux, the net K+ flow does not seem to involve the band 3 conformational change that mediates anion exchange. The data suggest that in low-Cl- media, the anion selectivity of capnophorin decreases so that it can act as a very low-conductivity channel for cations. Na+ and Rb+, as well as K+, can utilize this pathway.

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Treatment of human granulocytes with concanavalin A, phorbol myristate acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (FMLP), and A23187 (a calcium ionophore) stimulates the release of superoxide anion and the generation of chemiluminescence. The fluorescent probe, Di-S-C3(5), has been used to monitor shifts in membrane potential in response to these stimulants which precede the secretion of superoxide. Concanavalin A, PMA, and FMLP induce a biphasic shift in transmembrane potential (Em), i.e., a rapid depolarization followed by a prolonged hyperpolarization. This depolarization is dependent on both external sodium and calcium while the hyperpolarization is inhibited by ouabain which blocks the electrogenic Na-K pump. In contrast, A23187 induces a rapid and prolonged depolarization. This monophasic shift in Em is dependent on external calcium. These results suggest that depolarization acts as a signal to initiate events associated with the "respiratory burst" of these phagocytes.