June 2013
Volume 54, Issue 15
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ARVO Annual Meeting Abstract  |   June 2013
Effect of external divalent cations on Cx46 hemichannel activity
Author Affiliations & Notes
  • Lisa Ebihara
    Physiology, Rosalind Franklin Sch of Med, North Chicago, IL
  • Anna Rounis
    Physiology, Rosalind Franklin Sch of Med, North Chicago, IL
  • Jun-Jie Tong
    Physiology, Rosalind Franklin Sch of Med, North Chicago, IL
  • Footnotes
    Commercial Relationships Lisa Ebihara, None; Anna Rounis, None; Jun-Jie Tong, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3682. doi:
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      Lisa Ebihara, Anna Rounis, Jun-Jie Tong; Effect of external divalent cations on Cx46 hemichannel activity. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3682.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The lens has been proposed to have an internal microcirculation system that delivers nutrients to and remove waste products from the core of the lens. A key component of this system is a distributed sodium leak conductance. Here we investigate the hypothesis that Cx46 hemichannels may mediate the basal permeability of fiber cells to sodium observed in the intact lens.

Methods: Differentiating fiber cells were isolated from mouse lenses using collagenase. Membrane currents were recorded in fiber cells using whole cell patch clamping. Dye uptake was measured in transfected HeLa cells using time lapse imaging.

Results: KOCx50 fiber cells exhibited a large, nonselective cation current that was absent in fiber cells isolated from double KO (Cx50(-/-)Cx46(-/-)) mouse lenses confirming that it was due to Cx46 hemichannels. This current could be reversibly blocked by external calcium and magnesium in a concentration- and voltage-dependent manner. Calcium was a more potent blocker than magnesium. Cx46 hemichannels were largely closed at a resting voltage of -60 mV in the presence of physiological divalent cation concentrations (1 mM Ca2+, 1 mM Mg2+). However, even though the vast majority of these channels were closed at -60 mV, a small, persistent, nonselective cation current could still be detected using the whole cell patch clamp technique. This current could be blocked by increasing external calcium to 2 mM or by applying repetitive depolarizing steps to +30 mV and was not observed in fiber cells isolated from double KO mouse lenses suggesting that it was due to Cx46 hemichannels. To further investigate this phenomenon, we studied propidium iodide uptake in connexin transfected HeLa cells. In the presence of physiological divalent cation concentrations, HeLa cells expressing rCx46 showed significantly more propidium iodide uptake than parental HeLa cells or HeLa cells expressing other lens connexins such as rCx43 or chicken Cx50. Dye uptake by HeLa rCx46 cells could be greatly reduced by increasing Ca2+ to 5 mM suggesting that it was due to Cx46 hemichannels.

Conclusions: Our results suggest that Cx46 hemchannels may provide a novel pathway for influx of calcium and sodium under both physiological and pathological conditions.

Keywords: 445 cataract • 532 gap junctions/coupling  
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