ABSTRACT The M 2 ion channel of influenza A virus is a small integral membrane protein whose active form is a homotetramer with each polypeptide chain containing 96-amino-acid residues. To identify residues of the transmembrane (TM) domain that line the presumed central ion-conducting pore, a set of mutants was generated in which each residue of the TM domain (residues 25 to 44) was replaced by cysteine. The accessibility of the cysteine mutants to modification by the sulfhydryl-specific reagents methane thiosulfonate ethylammonium (MTSEA) and MTS tetraethylammonium (MTSET) was tested. Extracellular application of MTSEA evoked decreases in the conductances measured from two mutants, M 2 -A30C and M 2 -G34C. The changes observed were not reversible on washout, indicative of a covalent modification. Inhibition by MTSEA, or by the larger reagent MTSET, was not detected for residues closer to the extracellular end of the channel than Ala-30, indicating the pore may be wider near the extracellular opening. To investigate the accessibility of the cysteine mutants to reagents applied intracellularly, oocytes were microinjected directly with reagents during recordings. The conductance of the M 2 -W41C mutant was decreased by intracellular injection of a concentrated MTSET solution. However, intracellular application of MTSET caused no change in the conductance of the M 2 -G34C mutant, a result in contrast to that obtained when the reagent was applied extracellularly. These data suggest that a constriction in the pore exists between residues 34 and 41 which prevents passage of the MTS reagent. These findings are consistent with the proposed role for His-37 as the selectivity filter. Taken together, these data confirm our earlier model that Ala-30, Gly-34, His-37, and Trp-41 line the channel pore (L. H. Pinto, G. R. Dieckmann, C. S. Gandhi, C. G. Papworth, J. Braman, M. A. Shaughnessy, J. D. Lear, R. A. Lamb, and W. F. DeGrado, Proc. Natl. Acad. Sci. USA 94:11301–11306, 1997).
Double‐barrel micropipettes were used to pressure‐inject EGTA into the outer segments of rods in the isolated retina of Bufo marinus. We used these pipettes to point voltage clamp the cell to its resting membrane voltage during the injection of EGTA in order to prevent changes in membrane voltage from occurring. The input conductance of the rod was assessed by measuring the incremental membrane current required to hyperpolarize the membrane by less than or equal to 10 mV. When the retina was bathed in normal Ringer solution, the injection of EGTA during point voltage clamp evoked an inward membrane current and in increase in input conductance. This observation is consistent with an EGTA‐evoked increase in conductance for an ion with an equilibrium potential more depolarized than the resting membrane potential. Injections of control solutions that did not contain EGTA had no effect. The effects of injected EGTA were not altered by variations in the pH or buffering capacity of the injection solution, or by the addition of equimolar Mg2+. Furthermore, injections of a solution containing equimolar Ca2+ and EGTA were without effect. Thus, the observed effects of injected EGTA were due to the lowering of the [Ca2+]i. Replacement of extracellular Na+ with choline+ abolished both the response to light and the EGTA‐evoked increase in input conductance. A low [Na+]o solution containing 10(‐8) M‐Ca2+ reduced the response to injected EGTA by approximately the same amount as it reduced the response to light. Replacement of extracellular Cl‐ by methanesulphonate was without significant effect on either the response to light or to injected EGTA. These results are consistent with the interpretation that a lowered [Ca2+]i increases primarily the sodium conductance, gNa, of the plasma membrane of the rod outer segment. The conductance that is affected by a lowered [Ca2+]i appears to have the same specificity as the light‐dependent conductance. This conclusion is consistent with a hypothesis for visual transduction involving modulation of gNa by light‐evoked changes in the [Ca2+]i.
Amantadine inhibits the M2 proton channel of influenza A virus, yet most of the currently circulating strains of the virus carry mutations in the M2 protein that render the virus amantadine-resistant. While most of the research on novel amantadine analogues has revolved around the synthesis of novel adamantane derivatives, we have recently found that other polycyclic scaffolds effectively block the M2 proton channel, including amantadine-resistant mutant channels. In this work, we have synthesized and characterized a series of pyrrolidine derivatives designed as analogues of amantadine. Inhibition of the wild-type M2 channel and the A/M2-S31N, A/M2-V27A, and A/M2-L26F mutant forms of the channel were measured in Xenopus oocytes using two-electrode voltage clamp assays. Most of the novel compounds inhibited the wild-type ion channel in the low micromolar range. Of note, two of the compounds inhibited the amantadine-resistant A/M2-V27A and A/M2-L26F mutant ion channels with submicromolar and low micromolar IC50, respectively. None of the compounds was found to inhibit the S31N mutant ion channel.
The mutant mouse pearl, characterized by its hypopigmentation, has a specific functional defect in a sensory system—the retina. The intact pearl mouse has reduced sensitivity in the dark-adapted condition. Normal sensitivity is restored by isolation and superfusion of the retina with bicarbonate-buffered Ringer solution, suggesting that the retinal expression of the pearl mutation depends on a diffusible substance. The pearl phenotype is described as a possible model for human congenital stationary night blindness.
