Biochemical Requirements for Inhibition of Connexin26-containing Channels by Natural and Synthetic Taurine Analogs

Abstract

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...

Aminosulfonate Modulated pH-induced Conformational Changes in Connexin26 Hemichannels

“…No Conformational Change Occurs in the Absence of Aminosulfonates-According to previous findings (17,18), the pH-dependent gating of Cx26 could be only observed in the presence of aminosulfonate-containing compounds. To test this hypothesis and to prove that the conformational change observed can be indeed correlated to the gating mechanism, we imaged connexin preparations at different pH, buffered in nonaminosulfonate buffers.…”

“…One of the simplest of these aminosulfonate compounds is taurine, a naturally occurring ubiquitous cytoplasmic component (15,16). This pH sensitivity was directly attributed to binding of protonated aminosulfonates to Cx26, because homomeric Cx32 channels did not show this pH sensitivity (17,18). However, it should be noted that Cx46 hemichannels in excised patches appear to display pH sensitivity in the absence of any added cytosolic material (19).…”

Permeation of Calcium through Purified Connexin 26 Hemichannels

“…Sucrose Transport-Sucrose permeability of liposomes containing purified Cx26 was determined by the transport-specific fractionation technique (31)(32)(33). For these experiments, Cx26 was reconstituted in liposomes containing traces of phosphatidylethanolamine labeled with lissamine rhodamine B (PE-R; PC:PS:PE-R ratio of 2:1:0.05, w/w/w), using a buffer that contained 10 mM KCl, 0.1 mM EGTA, 459 mM urea, and 10 mM HEPES, pH 7.6.…”

“…Here, we performed functional studies to determine whether the reconstituted Cx26 has permeability properties that resemble those of Cx26 HCs. First, we determined whether reconstituted Cx26 HCs were permeable to sucrose using the transport-specific fractionation technique developed by Harris and co-workers (32,33) to separate sucrose-permeable from sucrose-impermeable liposomes (31). The method is based on the migration of liposomes upon centrifugation in a linear iso-osmolar sucrose gradient, with [sucrose] increasing from top to bottom, and a reversed urea gradient to maintain the osmolarity constant.…”

Cisplatin and Oxaliplatin Inhibit Gap Junctional Communication by Direct Action and by Reduction of Connexin Expression, Thereby Counteracting Cytotoxic Efficacy

“…These procedures were used according to previous protocols (Tao and Harris, 2004;He et al, 2009). After induction of connexin expression with doxycycline, cells were harvested, solubilized in n-octyl-␤-D-glucoside (Calbiochem, San Diego, CA), and the connexinpurified by immunoaffinity chromatography using a monoclonal antibody directed against the HA epitope tag (clone HA-7; SigmaAldrich, St. Louis, MO).…”