The connexin gene family codes for the protein subunits of gap junction channels that mediate direct diffusion of ions and metabolites between the cytoplasm of adjacent cells (reviewed by Bennett et al.

The female sex hormone estrogen (17b-estradiol; E2) may function as a neurohormone and has multiple neuromodulatory functions in the brain. Its potent neuroprotective activities can be dependent and independent of estrogen receptors (ERs). In addition, E2 influences the processing of the amyloid b precursor protein (APP), one central step in the pathogenesis of Alzheimer's disease. Here, we show: (a) that physiological concentrations of E2 very rapidly cause an increased release of secreted nonamyloidogenic APP (sAPPa) in mouse hippocampal HT22 and human neuroblastoma SK-N-MC cells; and (b) that this effect is mediated through E2 via the phosphorylation of extracellular-regulated kinase 1 and 2 (ERK1/2), prominent members of the mitogen-activated protein kinase (MAPK) pathway. Furthermore, we show that the activation of MAPK-signaling pathway and the enhancement of the sAPP release is independent of ERs and could be induced by E2 to a similar extent in neuronal cells either lacking or overexpressing a functional ER.

Our knowledge on functional aspects of gap junction channels has evolved quickly over the last decade. Progress has been closely associated with the availability of cDNAs coding for various gap junction proteins of vertebrates, the connexins. Expression studies with injected Xenopus oocytes and transfected mammalian cells have shown that the functional diversity of gap junctions reflects the structural variety of connexins (see Bruzzone et al. 1996). Recently, a new mouse connexin has been identified and cloned by screening of a mouse genomic library (Dahl et al. 1996). This protein has a molecular mass of 30•366 kDa and hence was called connexin30 (Cx30). Northern blots of total RNA from mouse tissues have indicated that Cx30 is strongly expressed in adult brain as well as skin and is less abundant in uterus, lung and eye tissue. Small amounts of transcripts have been detected in testis and sciatic nerve. No Cx30 mRNA has been found in liver. Sequence comparisons of nucleotides and amino acids suggest that mouse Cx30 is a close relative of mouse Cx26. There are few reports in the literature on functional aspects of Cx30 gap junctions. Injection of mouse Cx30 cRNA into Xenopus oocytes induced the formation of gap junctions susceptible to intercellular current flow and dye diffusion (Dahl et al. 1996). Neurobiotin diffusion experiments with transfected HeLa cells revealed that Cx30 forms heterotypic gap junction channels with Cx26, Cx30•3, Cx31, Cx40, Cx43, Cx45 and Cx50 (D. Manthey et al., in preparation). Electrophysiological studies on pairs of transfected HeLa

To evaluate the influence of intracellular domains of connexin (Cx) on channel transfer properties, we analyzed mouse connexin (Cx) Cx26 and Cx30, which show the most similar amino acid sequence identities within the family of gap junction proteins. These connexin genes are tightly linked on mouse chromosome 14. Functional studies were performed on transfected HeLa cells stably expressing both mouse connexins. When we examined homotypic intercellular transfer of microinjected neurobiotin and Lucifer yellow, we found that gap junctions in Cx30-transfected cells, in contrast to Cx26 cells, were impermeable to Lucifer yellow. Furthermore, we observed heterotypic transfer of neurobiotin between Cx30-transfectants and HeLa cells expressing mouse Cx30.3, Cx40, Cx43 or Cx45, but not between Cx26 transfectants and HeLa cells of the latter group. The main differences in amino acid sequence between Cx26 and Cx30 are located in the presumptive cytoplasmic loop and C-terminal region of these integral membrane proteins. By exchanging one or both of these domains, using PCR-based mutagenesis, we constructed Cx26/30 chimeric cDNAs, which were also expressed in HeLa cells after transfection. Homotypic intercellular transfer of injected Lucifer yellow was observed exclusively with those chimeric constructs that coded for both cytoplasmic domains of Cx26 in the Cx30 backbone polypeptide chain. In contrast, cells transfected with a construct that coded for the Cx26 backbone with the Cx30 cytoplasmic loop and C-terminal region did not show transfer of Lucifer yellow. Thus, Lucifer yellow transfer can be conferred onto chimeric Cx30 channels by exchanging the cytoplasmic loop and the C-terminal region of these connexins. In turn, the cytoplasmic loop and C-terminal domain of Cx30 prevent Lucifer yellow transfer when swapped with the corresponding domains of Cx26. In chimeric Cx30/Cx26 channels where the cytoplasmic loop and C-terminal domains had been exchanged, the unitary channel conductance was intermediate between those of the parental channels. Moreover, the voltage sensitivity was slightly reduced. This suggests that these cytoplasmic domains interfere directly or indirectly with the diffusivity, the conductance and voltage gating of the channels.

A new mouse connexin gene has been isolated that codes for a connexin protein of 505 amino acid residues. Based on the predicted molecular mass of 57.115 kDa, it has been designated connexin-57. Similar to most other mouse connexin genes, the coding region of connexin-57 is not interrupted by introns and exists in the mouse genome as a single-copy gene. Within the connexin family, this new gene shows highest sequence identity to porcine connexin-60 in the ␣ group of connexins. The connexin-57 gene was mapped to a position on mouse chromosome 4, 30 centimorgans proximal to a cluster of previously mapped connexin genes. Low levels of connexin-57 mRNA were detected in skin, heart, kidney, testis, ovary, intestine, and in the mouse embryo after 8