Browsing by Author "Reuss, Luis"
Now showing 1 - 5 of 5
Results Per Page
Sort Options
Item Biphasic effect of linoleic acid on connexin 46 hemichannels(2011) Retamal, Mauricio; Evangelista-Martínez, Flavio; León-Paravic, Carmen; Altenberg, Guillermo; Reuss, LuisConnexins form hemichannels at undocked plasma membranes and gap-junction channels (GJCs) at intercellular contacting zones. Under physiological conditions, hemichannels have low open probabilities, but their activation under pathological conditions, such as ischemia, induces and/or accelerates cell death. Connexin 46 (Cx46) is a major connexin of the lens, and mutations of this connexin induce cataracts. Here, we report the effects of linoleic acid (LA) on the electrical properties of Cx46 GJCs and hemichannels expressed in Xenopus laevis oocytes. LA has a biphasic effect, increasing hemichannel current at 0.1 mu M and decreasing it at concentrations of 100 mu M or higher. The effects of extracellular and microinjected LA conjugated to coenzyme A (LA-CoA) suggest that the current activation site is accessible from the intracellular but not extracellular compartment, whereas the current inhibitory site is either located in a region of the hemichannel pore inaccessible to intracellular LA-CoA, or requires crossing of LA through an organelle membrane. Experiments with other fatty acids demonstrated that the block of hemichannels depends on the presence of a hydrogenated double bond at position 9 and is directly proportional to the number of double bonds. Experiments in paired oocytes expressing Cx46 showed that LA does not affect GJCs. The block by unsaturated fatty acids reported here opens the possibility that increases in the concentration of these lipids in the lens induce cataract formation by blocking Cx46 hemichannels.Item Carbon monoxide (CO) is a novel inhibitor of connexin hemichannels(The American Society for Biochemistry and Molecular Biology, 2014) León-Paravic, Carmen; Figueroa, Vania; Guzmán, Diego; Valderrama, Carlos; Vallejos, Antonio; Fiori, Mariana; Altenberg, Guillermo; Reuss, Luis; Retamal, MauricioHemichannels (HCs) are hexamers of connexins that can form gap-junction channels at points of cell contacts or "free HCs" at non-contacting regions. HCs are involved in paracrine and autocrine cell signaling, and under pathological conditions may induce and/or accelerate cell death. Therefore, studies of HC regulation are of great significance. Nitric oxide affects the activity of Cx43 and Cx46 HCs, whereas carbon monoxide (CO), another gaseous transmitter, modulates the activity of several ion channels, but its effect on HCs has not been explored. We studied the effect of CO donors (CORMs) on Cx46 HCs expressed in Xenopus laevis oocytes using two-electrode voltage clamp and on Cx43 and Cx46 expressed in HeLa cells using a dye-uptake technique. CORM-2 inhibited Cx46 HC currents in a concentration-dependent manner. The C-terminal domain and intracellular Cys were not necessary for the inhibition. The effect of CORM-2 was not prevented by guanylyl-cyclase, protein kinase G, or thioredoxin inhibitors, and was not due to endocytosis of HCs. However, the effect of CORM-2 was reversed by reducing agents that act extracellularly. Additionally, CO inhibited dye uptake of HeLa cells expressing Cx43 or Cx46, and MCF-7 cells, which endogenously express Cx43 and Cx46. Because CORM-2 carbonylates Cx46 in vitro and induces conformational changes, a direct effect of that CO on Cx46 is possible. The inhibition of HCs could help to understand some of the biological actions of CO in physiological and pathological conditions.Item Cx46 hemichannel modulation by nitric oxide: Role of the fourth transmembrane helix cysteine and its possible involvement in cataract formation.(2019) Retamal, Mauricio; Orellana, Viviana; Arévalo, Nicolás; Rojas, Cristóbal; Arjona, Rodolfo; Alcaíno, Constanza; González, Wendy; Canan, Jonathan; Moraga-Amaro, Rodrigo; Stehberg, Jimmy; Reuss, Luis; Altenberg, GuillermoUnder normal conditions, connexin (Cx) hemichannels have a low open probability, which can increase under pathological conditions. Since hemichannels are permeable to relatively large molecules, their exacerbated activity has been linked to cell damage. Cx46 is highly expressed in the lens and its mutations have been associated to cataract formation, but it is unknown whether Cx46 has a role in non-genetic cataract formation (i.e. aging and diabetes). Nitric oxide (NO) is a key element in non-genetic cataract formation and Cx46 hemichannels have been shown to be sensitive to NO. The molecular mechanisms of the effects of NO on Cx46 are unknown, but are likely to result from Cx46 S-nitrosation (also known as S-nitrosylation). In this work, we found that lens opacity was correlated with Cx46 S-nitrosation in an animal model of cataract. Consistent with this result, a NO donor increased Cx46 S-nitrosation and hemichannel opening in HLE-B3 cells (cell line derived from human lens epithelial cells). Mutagenesis studies point to the cysteine located in the fourth transmembrane helix (TM4; human C212, rat C218) as the NO sensor. Electrophysiological studies performed in Xenopus oocytes revealed that rat Cx46 hemichannels are sensitive to different NO donors, and that the presence of C218 is necessary to observe the NO donors' effects. Unexpectedly, gap junctions formed by Cx46 were insensitive to NO or the reducing agent dithiothreitol. We propose that increased hemichannel opening and/or changes in their electrophysiological properties of human Cx46 due to S-nitrosation of the cysteine in TM4 could be an important factor in cataract formation.Item Functional hemichannels formed by human connexin 26 expressed in bacteria(© 2015 The Author(s) CC-BY Licence, 2015) Fiori, Mariana; Krishnan, Srinivasan; Cortés, Marien; Retamal, Mauricio; Reuss, Luis; Altenberg, Guillermo; Cuello, LuisGap-junction channels (GJCs) communicate the cytoplasm of adjacent cells and are formed by head-to-head association of two hemichannels (HCs), one from each of the neighbouring cells. GJCs mediate electrical and chemical communication between cells, whereas undocked HCs participate in paracrine signalling because of their permeability to molecules such as ATP. Sustained opening of HCs under pathological conditions results in water and solute fluxes that cannot be compensated by membrane transport and therefore lead to cell damage. Mutations of Cx26 (connexin 26) are the most frequent cause of genetic deafness and it is therefore important to understand the structure-function relationship of wild-type and deafness-associated mutants. Currently available connexin HC expression systems severely limit the pace of structural studies and there is no simple high-throughput HC functional assay. The Escherichia coli-based expression system presented in the present study yields milligram amounts of purified Cx26 HCs suitable for functional and structural studies. We also show evidence of functional activity of recombinant Cx26 HCs in intact bacteria using a new growth complementation assay. The E. coli-based expression system has high potential for structural studies and high-throughput functional screening of HCs.Item Voltage-dependent facilitation of Cx46 hemichannels(2010) Retamal, Mauricio A.; Yin, ShengYong; Altenberg, Guillermo A.; Reuss, LuisGap junction channels are formed by two hemichannels in series (one from each neighboring cell), which are in turn connexin hexamers. Under normal conditions, hemichannels at the plasma membrane are mostly closed but can be opened by changes in membrane voltage, extracellular divalent ion concentration, phosphorylation, pH, and redox potential. Recently, interactions between channels have been found to modulate the activity of several ion channels, including gap junction channels. Here, we studied whether connexin46 (Cx46) hemichannels display such behavior. We studied the response of the Cx46 hemichannels expressed in Xenopus laevis oocytes to consecutive depolarization pulses. Hemichannels formed by wild-type Cx46 and a COOH-terminal domain truncation mutant (Cx46ΔCT) were activated by voltage pulses. When the hemichannels were depolarized repeatedly from −60 mV to +80 mV, the amplitude of the outward and tail currents increased progressively with successive pulses. This phenomenon (“current facilitation”) depended on the amplitude of the depolarization, reaching a maximum at approximately +60 mV in oocytes expressing Cx46, and on the interval between pulses, disappearing with intervals longer than about 20 s. The current facilitation was also present in oocytes expressing Cx46ΔCT, ruling out a primary role of this domain in the facilitation. Nominal removal of divalent cations from the extracellular side caused maximal current activation of Cx46 and Cx46ΔCT hemichannels and prevented facilitation. The results suggest that Cx46 hemichannels show a cooperative activation independent of their COOH-terminal domain.