Binding Without Block. An Analysis of Amantadine and Rimantadine Block of the Influenza M2 S31N Channel

Abstract

The mutation from serine to asparagine at residue 31 (S31N) of the influenza A M2 channel is ubiquitous in the strains circulating in humans. This M2 mutant variant is insensitive to block by amantadine (AMT) and rimantadine (RMT), two previously FDA approved M2 blockers. It is unclear whether the inhibition results from weak binding or incomplete block. We addressed the question of “binding without block” using two-electrode voltage clamp (TEVC) electrophysiology. Data was collected in the wild-type (WT) and S31N M2 channels. Analysis of the electrophysiology data for complete or partial block by AMT and RMT was done using a one-site binding model nonlinear least squares curve fit with the to obtain rate constants. Results show that the S31N M2 channel is completely blocked by AMT at 10 mM, suggesting (proving?) there is no binding without block for AMT. In contrast, RMT achieves a modest maximal level of block in the S31N M2 channel at 1 mM, with very little increase in block at 10 mM. This suggests RMT binding has reached saturation and that there is binding without block. The rate constants obtained from the curve fit show a significant change in AMT or RMT and protein association. In both cases, the on rate constant has decreased whereas the off rate has increased in the S31N M2 variant compared to the rate constants in the WT M2 channel. These rate constants could be used to compare with molecular dynamics binding simulations to better understand the change in M2-AMT/RMT association.