A BSTR ACTDirect recruitment and activation of caspase-9 by Apaf-1 through the homophilic CARD͞CARD (Caspase Recruitment Domain) interaction is critical for the activation of caspases downstream of mitochondrial damage in apoptosis. Here we report the solution structure of the Apaf-1 CARD domain and its surface of interaction with caspase-9 CARD. Apaf-1 CARD consists of six tightly packed amphipathic ␣-helices and is topologically similar to the RAIDD CARD, with the exception of a kink observed in the middle of the N-terminal helix. By using chemical shift perturbation data, the homophilic interaction was mapped to the acidic surface of Apaf-1 CARD centered around helices 2 and 3. Interestingly, a significant portion of the chemically perturbed residues are hydrophobic, indicating that in addition to the electrostatic interactions predicted previously, hydrophobic interaction is also an important driving force underlying the CARD͞CARD interaction. On the basis of the identified functional residues of Apaf-1 CARD and the surface charge complementarity, we propose a model of CARD͞ CARD interaction between Apaf-1 and caspase-9.
In this work we examined the time course and the amount released, by hyposmolarity, for the most abundant free amino acids (FAA) in rat brain cortex astrocytes and neurons in culture. The aim was to evaluate their contribution to the process of cell volume regulation. Taurine, glutamate, and D-aspartate in the two types of cells, beta-alanine in astrocytes and GABA in neurons were promptly released by hyposmolarity, reaching a maximum within 1-2 min. after an osmolarity change. A substantial amount of the intracellular pool of these amino acids was mobilized in response to hyposmolarity. The amount released in media with osmolarity reduced from 300 mOsm to 150 mOsm or 210 mOsm, represented 50%-65% and 13%-31%, respectively, of the total amino acid content in cells. In both astrocytes and neurons, the efflux of glutamine and alanine was higher under isosmotic conditions and increased only marginally during hyposmotic conditions. 86Rb+, used as tracer for K+, was released from astrocytes, 30% and 11%, respectively, in hyposmotic media of 150 mOsm or 210 mOsm but was not transported in neurons. From these results it was calculated that FAA contribute 54% and inorganic ions 46% to the process of volume regulation in astrocytes exposed to a 150 mOsm hyposmotic medium. This contribution was 55% for FAA and 45% for K+ and Cl- in cells exposed to 210 mOsm hyposmotic solutions. These results indicate that the contribution of FAA to the process of cell volume regulation is higher in astrocytes than in other cell types including renal and blood cells.
The permeability of the hyposmolarity-activated pathway to amino acids and polyols in cultured astrocytes was examined following the change in rate and direction of regulatory volume decrease (RVD) when the extracellular concentration of the osmolytes was increased to reverse their intracellular-extracellular concentration gradient. Activation of the pathway by swelling would allow those permeable osmolytes to enter the cell and inhibit RVD. The pathway was found to be permeable to neutral amino acids, with beta-amino acids (beta-alanine = taurine > gamma-aminobutyric acid) more permeable than alpha-amino acids. Glycine, alanine, threonine, phenylalanine, and asparagine, but not glutamine, were permeable through this pathway. Aspartate was more permeable than glutamate, and K+ and not Na+ must be the accompanying cation. Basic amino acids were excluded. The dimension of the amino acid pore activated by hyposmolarity seems to be at the limit of glutamate-glutamine size. Influx rather than efflux of amino acids was observed when extracellular concentration was greater than intracellular concentration, with differences in the amount accumulated by cells correlating with their efficiency as RVD blockers. Influx of taurine (as representative of permeable amino acids) was inhibited by the Cl- channel blockers/exchangers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (40%) and dipyridamole (85%) , and it is suggested that amino acids permeate through an anion channel. Sorbitol and mannitol, but not inositol, exhibited a small inhibitory effect on the later phase of RVD, whereas inositol slightly accelerated RVD.
The effects of the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 1,9-dideoxyforskolin (DDF), dipyridamole, and niflumic acid and of the polyunsaturated fatty acids arachidonic, linolenic, and linoleic acids on regulatory volume decrease (RVD) and associated 125I and [3H]taurine fluxes in cultured rat cerebellar granule neurons were examined. Dose-response curves of NPPB, DDF, and dipyridamole showed 20-100% inhibition of RVD and osmolyte fluxes. Niflumic acid was less potent, requiring 150-600 microM to show effects of this magnitude. The polyunsaturated fatty acids (5-20 microM) inhibited 80-90% RVD and osmolyte fluxes, with arachidonic acid exhibiting the most potent effect. The volume-associated taurine efflux was somewhat higher in younger neurons, but the pharmacological sensitivity was essentially the same in immature and mature cells. The effects of all tested drugs on 125I and [3H]taurine fluxes were remarkably similar, indicating a close pharmacological sensitivity of the transport mechanism for the two osmolytes. This is in line with the suggestion of a common pathway for the volume-associated release of Cl and amino acids functioning as osmolytes.
The effects of hyposmotic conditions on taurine uptake and release were studied in mice cultured cerebellar granule cells. The effect of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) and of the divalent cations Mg++ and Mn++ on the hyposmolarity-induced changes in these parameters was investigated. Mg++ (20 mM) and Mn++ (5 mM) inhibited by 25% and 41%, respectively, the release of taurine observed in 30% hyposmolar media. DIDS (100 microM) inhibited this release by 46%. Taurine efflux evoked by 50% hyposmolar solutions was reduced about 40% by Mg++ and 55% by Mn++. Taurine uptake into the granule cells could be resolved into a high-affinity carrier-mediated component plus a nonsaturable diffusion component. The kinetic constants (Km and Vmax) for the high-affinity uptake were unaffected by a 50% decrease in the osmolarity. The diffusion constant for the nonsaturable taurine uptake was increased from 1.5 x 10(-4) in isosmotic media to 4.6 x 10(-4) ml x min-1 x mg-1 in hyposmotic (50%) media. This increase in the diffusional component of taurine uptake elicited by the hyposmotic condition was inhibited approximately 25% by either 100 microM DIDS or 5 mM Mn++. These results strongly suggest that the increase in taurine release induced by swelling under hyposmotic conditions is mediated by a diffusional process and not by a reversal of the high-affinity taurine carrier.
pICln has been proposed to be the swelling-activated anion channel responsible for ICl, swell, or a channel regulator. We tested the anion channel hypothesis by reconstituting recombinant pICln into artificial and biological membranes. Single channels were observed when pICln was reconstituted into planar lipid bilayers. In the presence of symmetrical 300 mM KCl, the channels had a high open probability and a slope conductance of 48 pS, and were outwardly rectifying. Reduction of trans KCl to 50 mM shifted the reversal potential by −31.2 ± 0.06 mV, demonstrating that the channel is at least seven times more selective for cations than for anions. Consistent with this finding, channel conductance was unaffected by substitution of Cl− with glutamate, but was undetectable when K+ was replaced by N-methyl-d-glucamine. Reconstitution of pICln into liposomes increased 86Rb+ uptake by three- to fourfold, but had no effect on 36Cl− uptake. Phosphorylation of pICln with casein kinase II or mutation of G54, G56, and G58 to alanine decreased channel open probability and 86Rb+ uptake. When added to the external medium bathing Sf9 cells, pICln inserted into the plasma membrane and increased cell cation permeability. Taken together, these observations demonstrate that channel activity is due to pICln and not minor contaminant proteins. However, these findings do not support the hypothesis that pICln is the anion-selective ICl, swell channel. The observed cation channel activity may reflect an as yet to be defined physiological function of pICln, or may be a consequence of in vitro reconstitution of purified, recombinant protein.
Purpose Conditional inactivation of connexin43 (Cx43) in the pigmented epithelium of the mouse eye results in a reduction in aqueous humor production and complete loss of the vitreous chamber. It was proposed that gap junctions between pigmented and nonpigmented epithelia of the ciliary body are critical for the production of the aqueous humor. To form such junctions, Cx43 in the pigmented epithelium must interact with connexin(s) present in the adjacent cells of the nonpigmented epithelium. The importance of Cx43 expression in the nonpigmented epithelium for the establishment of gap junctions and the regulation of intraocular pressure was tested. Methods To inactivate Cx43 in the nonpigmented epithelium of the mouse eye, a mouse line was crossed with a floxed Cx43 locus (Cx43flox/flox) and a transgenic mouse line expressing cre recombinase under the control of the Pax6α promoter. General eye structure was evaluated by light microscopy, gap junctions were analyzed by electron microscopy, and intraocular pressure was directly assessed with micropipettes. Results In Pax6α-cre/Cx43flox/flox mice, Cx43 was partially inactivated in the nonpigmented epithelium of the ciliary body and iris. Animals developed dilatations between the pigmented and nonpigmented epithelia and displayed a significant reduction in intraocular pressure. However, gap junctions between the ciliary epithelial layers were decreased but not eliminated. Conclusions Cx43 expression in the nonpigmented epithelium of the ciliary body contributes to the formation of gap junctions with the cells of the pigmented epithelium. These gap junctions play a critical role in maintaining the physical integrity of the ciliary body epithelium. Although the partial loss of Cx43 from the nonpigmented epithelium was correlated with a measurable drop in intraocular pressure, possible changes in Cx43 in the aqueous outflow pathway may provide an additional contribution to the observed phenotype.
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