Connexin Pore Block By ABG-Sugars

Abstract

Pore blockers are valuable for structure-function study of membrane channels. Prior work shows novel anthranilamide moieties (ABG) derivatized to maltosaccharides of different lengths (Gn: n-glucose) are size-indexed pore blockers of connexin channels: block occurs with size-match with a segment of the pore lumen, not if the lumen or blocker is too narrow or wide. Permeation studies using the same maltosaccharides derivatized with an uncharged fluorescent group (PA-sugars) show the narrowest part of the pore (size-selective filter) decreases Cx32>Cx26≈Cx26/Cx32, the last being heteromeric. Efficacy studies of ABG-sugar block from each side of the pore reveal new information about channel structure: connexin pores have constricted segments with which ABG-sugars interact, other than the size-selective filter. From the cytoplasmic side, ABG-G3 blocks Cx26 but not Cx32 or Cx26/Cx32 channels, and the wider ABG-G4 blocks with decreasing effectiveness Cx32>Cx26>Cx26/Cx32. From the extracellular side, ABG-G3 blocks Cx26/Cx32 better than Cx26, and does not block Cx32 channels, while ABG-G4 has no effect on any channels tested. If block were exclusively at the size-selective filter, the pattern of block from both sides of the pore should be identical and consistent with the PA-sugar pore sizing study. Instead, the data show that pore width varies as the selectivity filter is approached from one side or the other. Specifically, the pore lumen of homomeric Cx26 and Cx32 channels narrows on the cytoplasmic side of the selectivity filter. Intriguingly, heteromeric Cx26/Cx32 channels show unique and substantial narrowing extracellular to the selectivity filter. This is significant as most connexin channels in vivo are heteromeric, and heteromeric channels are selective amongst second messengers while the corresponding homomeric channels are not. Our data suggest a consequence of ‘heteromericity’ is that segment/s of a pore-lining domain are asymmetrically displaced toward the center of the lumen. Supported by GM36044, NS56509.