SCAM analysis of Panx1 suggests a peculiar pore structure

Vertebrates express two families of gap junction proteins: the well-characterized connexins and the pannexins. In contrast to connexins, pannexins do not appear to form gap junction channels but instead function as unpaired membrane channels. Pannexins have no sequence homology to connexins but are distantly related to the invertebrate gap junction proteins, innexins. Despite the sequence diversity, pannexins and connexins form channels with similar permeability properties and exhibit similar membrane topology, with two extracellular loops, four transmembrane (TM) segments, and cytoplasmic localization of amino and carboxy termini. To test whether the similarities extend to the pore structure of the channels, pannexin 1 (Panx1) was subjected to analysis with the substituted cysteine accessibility method (SCAM). The thiol reagents maleimidobutyryl-biocytin and 2-trimethylammonioethyl-methanethiosulfonate reacted with several cysteines positioned in the external portion of the first TM segment (TM1) and the first extracellular loop. These data suggest that portions of TM1 and the first extracellular loop line the outer part of the pore of Panx1 channels. In this aspect, the pore structures of Panx1 and connexin channels are similar. However, although the inner part of the pore is lined by amino-terminal amino acids in connexin channels, thiol modification was detected in carboxyterminal amino acids in Panx1 channels by SCAM analysis. Thus, it appears that the inner portion of the pores of Panx1 and connexin channels may be distinct

Structural and functional evolution of the P2Y12-like receptor group

Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y12-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y12, have been deorphanized. The P2Y12-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y12 in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y12-like receptor members

Structure-function analysis of connexin32

Individual cell-cell channels consist of two hemichannels, located on neighboring cell membranes, that are interconnected to form an hydrophilic pathway. Each hemichannel, or connexon, is made of six protein subunits, called connexins.Connexin32 liver gap junction protein has four transmembrane segments, two extracellular regions and three cytoplasmic segments, which include the amino and carboxyl termini.The process of cell-cell channel formation was investigated by altering specific amino acids in the presumed extracellular domains of the connexin32. It is these domains that must interact when two hemichannels dock to form an open cell-cell channel. The mutant connexins were generated by site-directed in vitro mutagenesis of a connexin32 cDNA. The mutated cDNAs were then transcribed in vitro and the mRNA was injected into Xenopus oocytes for expression. Junctional conductances between paired oocytes resulting from the expression of the mRNA were measured by the double-voltage clamp technique.Every amino acid replacement in connexin32 affected its channel-forming ability. Some replacements altered the docking specificity of the hemichannel. The replacement of any one of the six cysteines in the extracellular loops by serine yielded proteins incapable of making channels, indicating that the extracellular cysteines play a crucial role in channel formation.In addition, deletion mutants in the carboxyterminal domain of connexin32 were generated in order to determine the shortest segment still capable of forming channels. The transmembrane topology of some of these deletion mutants was investigated by in vitro translation in a reticulocyte lysate supplemented with canine microsomes.Net positive charge and length of the carboxyterminal segment were found to be determinants for channel formation. Phosphorylation of Ser233 and Ser240 was found not to be necessary for channel formation when expressed in Xenopus oocytes.Finally, connexin32 hexamer formation was studied by native polyacrylamide gel electrophoresis, sucrose gradients of the in vitro translation products combined with crosslinking reagents

Gating properties of connexin32 cell-cell channels and their mutants expressed in Xenopus oocytes

Carboxyl-terminal deletion mutants of the gap junction protein connexin32 were tested in the oocyte cell-cell channel assay. Oocytes expressing a mutant lacking 58 carboxyl terminal amino acids were found to exhibit junctional conductances of the same magnitude as oocytes expressing wild-type connexin32. The gating properties of the channels formed by this mutant of connexin32 with respect to transjunctional voltage and cytoplasmic acidification are indistinguishable from those found with wild-type connexin32 channels. This includes a novel pH-dependent voltage gate. In another mutant, two carboxyl terminal serine residues, Ser233 and Ser240, were replaced by Asn residues. This double mutant has properties indistinguishable from wild-type connexin32, suggesting that phosphorylation of either of these serines is not required for channel opening

Mutational analysis of gap junction formation.

The paired oocyte cell-cell channel assay was used to investigate the mechanisms involved in the process of formation of gap junction channels. Single oocytes, injected with connexin-specific mRNAs, accumulate a pool of precursors from which cell-cell channels can form rapidly upon pairing. Several lines of evidence, including immunohistochemistry and surface labeling, indicate that part of this precursor pool is located in the cell membrane, probably in the form of closed hemichannels. The homophilic binding of hemichannels to each other can be mimicked by synthetic peptides representing the extracellular loop sequences of connexin32. The peptides specifically suppress channel formation. A crucial role is established for the six cysteines in the extracellular domains that are conserved in all vertebrate gap junction proteins. Change of any of these cysteines into serines results in absolute loss of function of the mutant connexin. The effects of thiol-specific reagents on channel formation suggest that docking and/or opening of channels involves disulfide exchange. Several of the variable amino acids in the extracellular loop sequences were found to determine specificity of connexin-connexin interactions

Formation of hybrid cell-cell channels.

The oocyte cell-cell channel assay was used to demonstrate that connexin-43 is a cell-cell channel-forming protein as previously shown for connexin-32. Expression of connexin-32 in one and connexin-43 in the other oocyte of a pair results in the formation of junctional conductances at rates similar to those observed when only one or the other connexin is expressed in both oocytes of a pair. This suggests that hybrid cell-cell channels form in the oocyte system. Hybrid channels also form when a connexin-43 mRNA-injected oocyte is paired with a noninjected oocyte expressing endogenous connexin. The latter hybrids have properties apparently contributed by both types of hemichannels. Pure connexin-43 channels are not voltage gated, whereas pure oocyte channels are voltage dependent; hybrids of these channels rectify

Cell/cell channel formation involves disulfide exchange

The oocyte cell/cell‐channel assay was used to identify amino acids involved in the process of cell/cell‐channel formation. The expression of the rat liver gap‐junction protein, connexin 32, in single oocytes, results in the accumulation of a pool of channel precursors. Upon pairing of such oocytes, cell/cell channels form rapidly from this pool. The rate of formation is affected by thiol‐specific reagents and the pH. This suggests the involvement of extracellular cysteine residues in the channel formation process. Two connexin‐32 mutants were generated by site‐directed mutagenesis in which cysteine residues were replaced by serine. Both mutant connexins were unable to form cell/cell channels. Thus, the cysteine residues appear to play an important role in the channel formation process