Previous work has shown that protonated taurine and aminosulfonate pH buffers, including HEPES, can directly and reversibly inhibit connexin channels that contain connexin26 (Cx26) (Bevans, C. G., and Harris, A. L. (1999) J. Biol. Chem. 274, 3711-3719). The structural requirements for this inhibition were explored by studies of the effects of structural analogs of taurine on the activity of Cx26-containing reconstituted hemichannels from native tissue. Several analogs inhibited the channels, with a range of relative affinities and efficacies. Each active compound contains a protonated amine separated from an ionized sulfonate or sulfinate moiety by several methylene groups. The inhibition is eliminated if the sulfonate/sulfinate moiety or the amine is not present. Compounds that contain a protonated amine but lack a sulfonate/sulfinate moiety do not inhibit but do competitively block the effect of the active compounds. Compounds that lack the protonated amine do not significantly inhibit or antagonize inhibition. The results suggest involvement of the protonated amine in binding and of the ionized sulfur-containing moiety in effecting the inhibition. The maximal effect of the inhibitory compounds is enhanced when a carboxyl group is linked to the ␣-carbon. Inhibition but not binding is stereospecific, with L-isomers being inhibitory and the corresponding D-isomers being inactive but able to antagonize inhibition by the L-isomers. Whereas not all connexins are sensitive to aminosulfonates, the well defined structural requirements described here argue strongly for a highly specific regulatory interaction with some connexins. The finding that cytoplasmic aminosulfonates inhibit connexin channels whereas other cytoplasmic compounds antagonize the inhibition suggests that gap junction channels are regulated by a complex interplay of cytoplasmic ligands.Connexin channels, which compose most gap junctions in vertebrates, mediate direct intercellular movement of cytoplasmic signaling molecules. There are ϳ20 isoforms of connexin protein (2), each of which forms channels with distinct regulatory and permeability properties (3). The intercellular signaling mediated by connexin channels is important; every functional deletion of a connexin isoform produces a distinct pathology (2). The pathologies that arise from altered connexin channel function must arise from abnormal molecular movement through connexin channels, whether in modulation, magnitude, or molecular specificity.Despite the importance of gap junction channels in development, physiology, and disease, little is known about the regulation of connexin channels by cytoplasmic ligands. Identification of endogenous ligands and their modes of action on connexin channels would be of considerable value for understanding intercellular signaling and connexin channel structure function.Connexin channels are homo-or hetero-oligomers of isoforms of connexin protein (4 -8). They have two functional and structural forms. The basic unit is a hexamer, called a "hemichannel" or "c...