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L. Ebihara, J.-J. Tong, T.-L. Chen, B. M. Vertel; Cx46 Forms Functional Hemichannels in Freshly Dissociated Mouse Fiber Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3463. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize the properties of Cx46 hemichannels in differentiating fiber cells isolated from mouse lenses.
Differentiating fiber cells were isolated from mouse lenses using collagenase. Cellular localization of Cx50 and Cx46 was assessed by immunofluorescence. Membrane currents were recorded using whole cell patch clamping. Cesium was used as the main cation in the pipette solution to block outward potassium currents. Chloride was replaced by gluconate in the bath solution to eliminate outward currents due to chloride influx. Dye uptake was measured using time lapse imaging.
In freshly dissociated fiber cells isolated from KOCx50 mouse lenses, removal of external divalent cations induced a macroscopic current composed of large conductance channels. This current was partially closed at a holding potential of -60 mV, activated on depolarization and had a reversal potential near 0 mV. These properties were very similar to those of Cx46 hemichannel currents recorded in single Xenopus oocytes. The amplitude of the current was linearly correlated to cell length. If the currents observed in divalent cation-free Ringer’s solution are due to Cx46 hemichannel opening, then dye influx by gap junctional/hemichannel permeable dyes should be measurable in the fiber cells. To measure dye influx, we used the positively charged dyes, propidium iodide (PrI) and DAPI. In the absence of external calcium, fiber cells took up both dyes. Furthermore, dye influx could be prevented by incubating the fiber cells in the presence of hemichannel blockers such as calcium, gadolinium, octanol, heptanol, and FFA. To confirm that this current was due to Cx46 hemichannels, we studied fiber cells isolated from the lenses of double knockout (Cx46 (-/-); Cx50(-/-)) mice and demonstrated that both the calcium-sensitive conductance and dye influx were absent.
These results show that Cx46 can form functional hemichannels in the nonjunctional membrane of fiber cells.
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