Connexin 35 (Cx35) is a major component of electrical synapses in the central nervous system. Many gap junctions containing Cx35 are regulated by dopamine receptor pathways that involve protein kinase A (PKA). To study the mechanism of PKA regulation, we analyzed direct phosphorylation of Cx35 by PKA in vitro, and studied the regulation of Neurobiotin tracer coupling in HeLa cells expressing Cx35 or Cx35 mutants that lack phosphorylation sites. In Cx35-transfected cells, application of the PKA activator Sp-8-cpt-cAMPS caused a significant decline in coupling, while a PKA inhibitor, Rp-8-cpt-cAMPS, significantly increased tracer coupling. In vitro phosphorylation and mutagenic analysis showed that PKA phosphorylates Cx35 directly at two major sites, Ser110 in the intracellular loop and Ser276 in the carboxyl terminus. In addition, a minor phosphorylation site in the C-terminus was identified by truncation of the last 7 amino acids at Ser298. The mutations Ser110Ala or Ser276Ala significantly reduced regulation of coupling by the PKA activator, while a combination of the two eliminated regulation. Truncation at Ser298 reversed the regulation such that the PKA activator significantly increased and the PKA inhibitor significantly decreased coupling. The activation was eliminated in the S110A,S276A,S298ter triple mutant. We conclude that PKA regulates Cx35 coupling in a complex manner that requires both major phosphorylation sites. Furthermore, the tip of the C-terminus acts as a "switch" that determines whether phosphorylation will inhibit or enhance coupling. Reliance on the combined states of three sites provides fine control over the degree of coupling through Cx35 gap junctions.

We examined the interactions of calmodulin with neuronal gap junction proteins connexin35 (Cx35) from perch, its mouse homologue Cx36, and the related perch Cx34.7 using surface plasmon resonance. Calmodulin bound to the C-terminal domains of all three connexins with rapid kinetics in a concentration- and Ca2+-dependent manner. Dissociation was also very rapid. K(d)'s for calmodulin binding at a high-affinity site ranged from 11 to 72 nM, and K(1/2)'s for Ca2+ were between 3 and 5 microM. No binding to the intracellular loops was observed. Binding competition experiments with synthetic peptides mapped the calmodulin binding site to a 10-30 amino acid segment at the beginning of the C-terminal domain of Cx36. The micromolar K(1/2)'s and rapid on and off rates suggest that this interaction may change dynamically in neurons, and may occur transiently when Ca2+ is elevated to a level that would occur in the near vicinity of an activated synapse.

Gap-junctional coupling among neurons is subject to regulation by a number of neurotransmitters including nitric oxide. We studied the mechanisms by which NO regulates coupling in cells expressing Cx35, a connexin expressed in neurons throughout the central nervous system. NO donors caused potent uncoupling of HeLa cells stably transfected with Cx35. This effect was mimicked by Bay 21-4272, an activator of guanylyl cyclase. A pharmacological analysis indicated that NOinduced uncoupling involved both PKG-dependent and PKG-independent pathways. PKA was involved in both pathways, suggesting that PKG-dependent uncoupling may be indirect. In vitro, PKG phosphorylated Cx35 at three sites: Ser110, Ser276 and Ser289. A mutational analysis indicated that phosphorylation on Ser110 and Ser276, sites previously shown also to be phosphorylated by PKA, had a significant influence on regulation. Ser289 phosphorylation had very limited effects. We conclude that NO can regulate coupling through Cx35 and that regulation is indirect in HeLa cells.

Gap junctions formed of connexin 36 (Cx36, also known as Gjd2) show tremendous functional plasticity on several time scales. Changes in connexin phosphorylation modify coupling in minutes through an order of magnitude, but recent studies also imply involvement of connexin turnover in regulating cell-cell communication. We utilized Cx36 with an internal HaloTag to study Cx36 turnover and trafficking in cultured cells. Irreversible, covalent pulse-chase labeling with fluorescent HaloTag ligands allowed clear discrimination of newly formed and pre-existing Cx36. Cx36 in junctional plaques turned over with a half-life of 3.1 h, and the turnover rate was unchanged by manipulations of protein kinase A (PKA) activity. In contrast, changes in PKA activity altered coupling within 20 min. New Cx36 in cargo vesicles was added directly to existing gap junctions and newly made Cx36 was not confined to points of addition, but diffused throughout existing gap junctions. Existing connexins also diffused into photobleached areas with a half-time of less than 2 s. In conclusion, studies of Cx36-HaloTag revealed novel features of connexin trafficking and demonstrated that phosphorylation-based changes in coupling occur on a different time scale than turnover.

[3H]Serotonin is accumulated by a specific set of amacrine cells in the rabbit retina. These cells also accumulate the neurotoxin, 5,7-dihydroxytryptamine, and show signs of necrosis within 4 h of in vivo exposure to the drug. Biochemical analysis of [3H]serotonin uptake reveal a sodium-and temperature-dependent, high affinity uptake system with a Km of 0.94 #M and Vmax of 1.08 pmol/mg protein/min. [3H]Tryptophan is also accumulated in rabbit retinal homogenates by a high affinity process. Accumulated [3H]serotonin is released in response to potassium-induced depolarization of intact, isolated retinas. In vitro binding studies of rabbit retinal homogenate membranes demonstrate specific sets of binding sites with characteristics of the postsynaptic serotonin receptor. These data strongly suggest that rabbit retina contains virtually all of the molecular components required for a functional serotonergic neurotransmitter system. The only significant difference between the serotonin system in rabbit retina and that in the well-established serotonin transmitter systems in nonmammalin retinas and in brains of most species is the relatively low concentration of endogenous serotonin in rabbit retinas, as demonstrated by high-performance liquid chromatography, histofluorescence, or immunocytochemistry.Serotonin has been identified as a transmitter in nervous tissue from the brain and retina of many vertebrates. Retinas from frogs and goldfish contain relatively high levels of endogenous serotonin (14,22), while bird and lizard retinas contain somewhat lower levels (7,15). In these species, neurons that possess endogenous stores of serotonin (demonstrated by histofluorescence or immunocytochemical techniques) and/or serotonin uptake systems (demonstrated by histofluorescence, autoradiography, or neurotoxic reactions) have neurites that are limited to the inner plexiform layer. The cell bodies of these neurons are found primarily in the amacrine cell layer of the inner nuclear layer, although some are found in the ganglion cell layer and are thought to be displaced amacrine cell bodies. One exception is the report by Tornquist et al. (22) that describes accumulation of [3H]-serotonin by cells in the outer plexiform layer of pigeon and chick retinas, tentatively designated interplexiform cells.Although serotonin is considered a strong transmitter candidate in the retinas of the nonmammalian species mentioned above, its role in mammalian retinas has been questioned because of the relatively low concentration of endogenous serotonin. Consistent with this finding, attempts to demonstrate endogenous serotonin by histofluorescence or immunocytochemistry have failed. Thus, Ehinger et al. (4) (see also reference 7) have proposed that the true indoleamine transmitter is not serotonin but some other closely related compound. By using a microdansylation procedure, Osborne et al. (10,12) observed that the low concentrations of serotonin in the bovine retina were localized in the inner nuclear and inner plexiform layers that ...