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A. Narayanasamy, K. Coral, M. Rahman, C. Nagaraj, R. Suryaraja, C. Madhavan, S. V Ramanan; Existence and Properties of Heteromeric Gap Junction Channels in Retinal Pigment Epithelial (RPE) Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5545.
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To characterize the properties of gap junctions in Bovine Retinal Pigment epithelial (RPE) cells.
Cultured primary Bovine RPE cells were well coupled by double whole cell patch clamp, with conductances ranging predominantly from 0-25 nS (mean = 8 nS), using a pulse protocol from -120mV to + 120mV for 200ms which elicited voltage dependent junctional currents in the bovine RPE. Longer pulses (20 s) as well as long ramps (20 s) were also used to observe slow kinetics. Western blotting and immunostaining was done to detect Connexin-43 (Cx43) and Connexin-50 (Cx50). Carboxyfluorescein dye (MW 376) permeability across the gap junctional channels as observed by optical monitoring of the probe flow served as an index of macromolecular transfer.
Both Western blotting and immunostains showed the presence of Cx43, but Cx50 was detected in Western blots alone. Pulsing RPE cells with brief (200 ms) voltage steps showed that the fast kinetics of the channel were symmetrically dependent on the transjunctional potential. However, with longer pulses, the channel showed marked asymmetry with voltage polarity, especially when the coupling was moderate or low (1-5 nS). In many asymmetric records, the gating charge for one polarity was as high as 10, consistent with a Cx50 component dominating heteromeric channel gating behavior. However, the unitary conductance of the channel was not polarity-dependent, with 80 pS being the most common unitary transition. Carboxyfluorescein has a high (0.1 relative to CsCl) permeability across gap junctional channels. A Poisson-like distribution of junctional conductances and dye permeabilities indicated that channels arrive and leave the junctional area in clusters, with about 100 channels in a cluster. This number is consistent with the size of endo- and exocytotic vesicles associated with gap junctional plaques that has been reported earlier.
The bovine retinal pigment epithelial cells are highly coupled by gap junctional channels. These channels possess properties not only similar to Cx43 channels, but also to that of Cx50 channels.
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