Connexin channels are a family of non-beta-barrel large-pore channels permeable to cellular signaling molecules. They are involved in a myriad of important physiological and pathophysiological processes in eukaryotes. However, the mechanisms of the large-pore channel molecular selectivity remain unclear. Here <b><i>we report the thermodynamics and kinetics of binding and transport of an important biological signaling molecule – cAMP - through Cx26 hemichannel </i></b>using atomistic simulations under voltages and Voronoi-tessellated Markovian milestoning. The inward and outward fluxes of cAMP were first obtained from 4 μs simulations at opposite voltages with multiple cAMPs in solution. The results are compared with the intrinsic potential of mean force (PMF) and the mean first passage times (MFPTs) of a single cAMP in the absence of voltage, obtained from a total of 16.5 μs of multi-replica Voronoi-tessellated Markovian milestoning simulations. Both voltage simulations and milestoning simulations revealed two cAMP binding sites. PMF and MFPTs were used to estimate binding constants and dissociation rates. We found that the dipole inside the pore produces an asymmetric PMF, reflected in unequal MFPTs in each direction at zero voltage. The free energy profiles under membrane potential were derived from intrinsic PMF and revealed the unique role of channel polarity and the mobile electrolyte within the pore on the total free energy. In addition, we show how these factors influence the cAMP dipole vector during permeation, and how cAMP affects the local and non-local pore diameter in a position-dependent manner. The computational approaches presented in this study can be applied to understand molecular permeation through large pores in general.

Asymmetric free energy and kinetics of cAMP permeation through connexin hemichannel

ACS Spring Meeting 2021

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