Connexins are the protein subunits of gap junction channels that allow a direct signaling pathway between networks of cells. The speci¢c role of connexin channels in the homeostasis of di¡erent organs has been validated by the association of mutations in several human connexins with a variety of genetic diseases. Several connexins are present in the mammalian cochlea and at least four of them have been proposed as genes causing sensorineural hearing loss. We have started our functional analysis by selecting nine mutations in Cx26 that are associated with non-syndromic recessive deafness (DFNB1). We have observed that both human Cx26 wild-type (HCx26wt) and the F83L polymorphism, found in una¡ected controls, generated electrical conductance between paired Xenopus oocytes, which was several orders of magnitude greater than that measured in water-injected controls. In contrast, most recessive Cx26 mutations (identi¢ed in DFNB1 patients) resulted in a simple loss of channel activity. In addition, the V37I mutation, originally identi¢ed as a polymorphism in heterozygous una¡ected individuals, was devoid of function and thus may be pathologically signi¢-cant. Unexpectedly, we have found that the recessive mutation V84L retained functional activity in both paired Xenopus oocytes and transfected HeLa cells. Furthermore, both the magnitude of macroscopic junctional conductance and its voltage-gating properties were indistinguishable from those of HCx26wt. The identi¢cation of functional di¡erences of disease causing mutations may lead to de¢ne which permeation or gating properties of Cx26 are necessary for normal auditory function in humans and will be instrumental in identifying the molecular steps leading to DFNB1. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
Glial calcium signals play important roles during CNS development. Calcium transients induced by ATP, acting on purinergic receptors, stimulate DNA synthesis, increase astrocytic and neural stem cell proliferation, and are prominent during the differentiation of radial glia. We have shown previously that expression of P2Y receptors in astrocytes is altered when connexin43 (Cx43) is downregulated. To evaluate the consequences of Cx43 deletion on calcium signaling during neural progenitor development, studies were performed on neurospheres derived from embryonic striatum. After adhesion, cells migrating from wild-type (WT) and Cx43-null neurospheres displayed spontaneous calcium oscillations. Such activity was blunted by apyrase, 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS-2179), and suramin, suggesting that ATP released by neural cells acts on purinergic receptors to induce calcium oscillations. The amplitudes of Ca2+ transients induced by P2Y but not P2X receptor agonists were larger in WT than in Cx43-null progenitors, suggesting that these two cell populations express different P2 receptors. Suramin, a nonselective P2 receptor antagonist, and MRS-2179, a P2Y1 receptor-selective antagonist, reduced the proliferation rate and the migration of WT progenitor cells to levels similar to those of Cx43-null cells. Conversely, exogenous expression of P2Y1 receptors in Cx43-null cells restored their migration pattern to levels seen in WT progenitors. However, treatment with P2 receptor antagonists did not alter the ratio of nestin to GFAP expression in WT neural progenitors. These data show that altered autocrine-paracrine communication attributable to reduced levels of P2Y1 receptors in neural progenitor cells lacking Cx43 affects proliferation and migration but not cell differentiation during early CNS development.
The asymmetric forms of cholinesterases are synthesized only in differentiated muscular and neural cells of vertebrates. These complex oligomers are characterized by the presence of a collagen-like tail, associated with one, two or three tetramers of catalytic subunits. The collagenic tail is responsible for ionic interactions, explaining the insertion of these molecules in extraceliular basal lamina, e.g. at neuromuscular endplates. We report the cloning of a collagenic subunit from Torpedo marmorata acetylcholinesterase (AChE). The predicted primary structure contains a putative signal peptide, a proline-rich domain, a collagenic domain, and a C-terminal domain composed of proline-rich and cysteine-rich regions. Several variants are generated by alternative splicing. Apart from the collagenic domain, the AChE tail subunit does not present any homology with previously known proteins. We show that coexpression of catalytic AChE subunits and collagenic subunits results in the production of asymmetric, collagen-tailed AChE forms in transfected COS cells. Thus, the assembly of these complex forms does not depend on a specific cellular processing, but rather on the expression of the collagenic subunits.
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