pH-induced closure of connexin43 (Cx43) channels involves interaction of the Cx43 carboxyl-terminal (Cx43CT) with a separate "receptor" domain. The receptor location and structure and whether the interaction is directly intramolecular are unknown. Here we show resonant mirror technology, enzyme-linked sorbent assays, and nuclear magnetic resonance (NMR) experiments demonstrating pH-dependent binding of Cx43CT to region 119 -144 of Cx43 (Cx43L2), which we propose is the receptor. NMR showed that acidification induced ␣-helical order in Cx43L2, whereas only a minor modification in Cx43CT structure was detected. These data provide the first demonstration of chemically induced structural order and binding between cytoplasmic connexin domains.

Abstract-Ischemia-induced acidification of astrocytes or cardiac myocytes reduces intercellular communication by closing gap junction channels and subsequently internalizing gap junction proteins. To determine whether such coupling changes might be attributable to altered interactions between connexin43 (Cx43) and other proteins, we applied the nigericin/high K ϩ method to vary intracellular pH (pHi) in cultured cortical astrocytes. Intracellular acidification was accompanied by internalization of Cx43 with retention of Cx43 scaffolding protein Zonula Occludens-1 (ZO-1) at cell surfaces, suggesting that ZO-1 and Cx43 dissociate at low pHi. Coimmunoprecipitation studies revealed decreased binding of ZO-1 and increased binding of c-Src to Cx43 at low pHi. Resonant mirror spectroscopy was used to quantify binding of the SH3 domain of c-Src and the PDZ domains of ZO-1 to the carboxyl terminal domain of Cx43 (Cx43CT). Data indicate that the c-Src/Cx43CT interaction is highly pH dependent whereas the ZO-1/Cx43CT interaction is not. Moreover, binding of c-Src to Cx43CT prevented and reversed ZO-1/Cx43CT binding. We hypothesize that increased affinity of c-Src for Cx43 at low pHi aids in separation of Cx43 from ZO-1 and that this may facilitate internalization of Cx43. These data suggest that protracted acidification may remodel protein-protein interactions involving Cx43 and thus provide an important protective mechanism to limit lesion spread after ischemic injury. Key Words: gap junctions Ⅲ ischemia Ⅲ protein-protein interactions Ⅲ resonant mirror spectroscopy Ⅲ central nervous system C onnexins are membrane proteins that oligomerize to form gap junction channels, through which ions and small molecules diffuse between cells. The present view is that these channels are not passive pores insulated from the rest of the molecular environment but rather are dynamic protein complexes that can both filter and amplify passage of information between cells. Among proteins known to associate with Connexin43 (Cx43) (the most abundant gap junction in heart and in astrocytes 1 ) are the scaffolding protein Zonula Occludens-1 (ZO-1) and the tyrosine kinase c-Src. [2][3][4][5] Recent studies show that this association is potentially interlinked, because binding of Cx43 to constitutively active c-Src is correlated with a decrease in the Cx43/ZO-1 interaction. 4,5 Still unclear are whether there is a direct interdependence to these interactions and whether these associations are modified by factors that close gap junctions. The present study centers on the pH dependence of interactions of Cx43 with ZO-1 and c-Src.Intracellular acidification leads to closure of gap junction channels, 6,7 which is relevant in the context of ischemia, where a drop in intracellular pH (pHi) has been demonstrated both in heart 8 and in brain. 9 Cardiac ischemia leads to electrical uncoupling 10 as well as to dephosphorylation and internalization of Cx43. 11 Changes in the composition of gap junction complexes attributable to sustained intracellular acidifi...

Norepinephrine is a stress hormone that enhances bacterial growth. We examined the effects of a small inoculum on the norepinephrine-induced growth of species previously reported to be unaffected by norepinephrine. The results indicated that a reduced inoculum density is essential for observing norepinephrineinduced effects. Additional studies using serum-free media suggested that transferrin plays a role in norepinephrine-induced growth.Determining the direct effect of catecholamines on the in vitro growth response of bacteria is one interdisciplinary approach that has been utilized to increase our understanding of the role that stress hormones play in the establishment and progression of infection in a host (21). Although a great deal of evidence suggests that stress-induced neurohormones play a critical role in the outcome of infections (1,3,4,5,7,21,31), the mechanisms by which these hormones act in the host remain unclear. Studies with human and animal models have indicated that increased levels of stress hormones, including norepinephrine (NE), as well as other catecholamines, alter the immune response and physiology of the host (2). High circulatory levels of these hormones are detected in individuals exposed to a variety of physically and/or mentally stressful situations, including trauma, space flight, and sepsis (13,29,31,32). The increases not only may alter the immune function but also may contribute to host morbidity and increased risk of infection (1,7,34). In the current study we reexamined the in vitro growth responses of a variety of bacterial species that were previously tested and reported not to be enhanced by the addition of NE (6,8).The conditions employed in this study include a minimally nutritive low-iron medium previously shown to maintain bacteriostasis (21), a low initial inoculum density of bacteria (10 CFU/ml) in order to capture the lag phase of the bacterial growth curve typically observed in a bacteriostatic medium (21,26), and a concentration of NE (0.0001 M) (14, 15, 26) which corresponds to target tissue levels and not mere plasma spillover (16,18,20). These rigorous conditions better represent in vivo milieus and also allow more suitable evaluation of a growth enhancement effect without the camouflage of rapid bacterial growth encountered when rich medium and large inocula are employed.Using a lower initial inoculum density (approximately 10 CFU/ml), each species tested exhibited NE-induced enhancement of in vitro growth compared to nontreated controls. Cultures of Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, Shigella sonnei, and Staphylococcus aureus grown in the presence of NE had shortened lag times and exhibited significant increases in bacterial counts (CFU/ml) at 18 and 24 h (Fig. 1) compared to the control. Moreover, for all of the gram-negative pathogens there were other times when there were significant increases in growth (Fig. 1A to D). S. aureus growth was only moderately affected by NE treatment, and there were significant differences in g...

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