Properties of connexin 46 hemichannels in dissociated lens fiber cells.
OBJECTIVE
To characterize the properties of connexin 46 hemichannels in differentiating fiber cells isolated from mouse lenses.


METHODS
Differentiating fiber cells were isolated from mouse lenses using collagenase. Cellular localization of connexin 50 (Cx50) and connexin 46 (Cx46) was assessed by immunofluorescence. Membrane currents were recorded using whole cell patch clamping. Dye uptake was measured using time-lapse imaging.


RESULTS
In freshly dissociated fiber cells isolated from knockout Cx50 (KOCx50) mouse lenses, removal of external divalent cations induced a macroscopic current composed of large conductance channels. This current was reduced 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. If the currents observed in divalent cation-free Ringer's solution were 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, the authors used the positively charged dyes, propidium iodide (PrI) and 4'-6-diamidino-2-phenylindole (DAPI). In the absence of external calcium, fiber cells took up both dyes. Furthermore, dye influx could be inhibited by hemichannel blockers. To confirm that this current was due to Cx46 hemichannels, the authors 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.


CONCLUSIONS
These results show that Cx46 can form functional hemichannels in the nonjunctional membrane of fiber cells.
Ebihara
Lisa
L
Departments of Physiology and Biophysics, Rosalind Franklin School of Medicine and Science, North Chicago, Illinois 60064, USA. lisa.ebihara@rosalindfranklin.edu
Tong
Jun-Jie
JJ
Vertel
Barbara
B
White
Thomas W
TW
Chen
Tung-Ling
TL
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D017630:Connexins / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D007908:Lens, Crystalline / Q000166:cytology* / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D018408:Patch-Clamp Techniques; D011419:Propidium / Q000378:metabolism; D059008:Time-Lapse Imaging; D000099264:Connexin 50
D017630:Connexins; D005136:Eye Proteins; D005456:Fluorescent Dyes; D007211:Indoles; D007473:Ion Channels; D011419:Propidium; D000099264:Connexin 50; C082917:GJA3 protein, human; C007293:DAPI