Properties of whole-cell ionic currents in cultured human corneal epithelial cells

Charles S. Bockman, May Griffith, Mitchell Aaron Watsky

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

PURPOSE. To identify and partially characterize the ionic currents contributing to the whole-cell conductance of cultured human corneal epithelial cells. METHODS. Epithelial cells were scraped from human donor corneas and cultured for use in patch-clamp experiments. Amphotericin B and the perforated-patch configuration were used to measure whole-cell currents in cells isolated from confluent monolayers. RESULTS. Cell monolayers exhibited cobblestone morphology and were immunopositive for corneal epithelium-specific cytokeratin. Single cells had a capacitance of 21 ≠ 2 pF and expressed similar types of ionic currents regardless of passage number. In descending order of frequency of occurrence, cells exhibited a nonselective cation current active at depolarized voltages and insensitive to Ba2+ and Gd3+; an outwardly rectifying K+ current active at depolarized voltages, stimulated by flufenamic acid and inhibited by tetraethylammonium; a voltage-gated inward Na+ current; an outwardly rectifying K+ current active at hyperpolarized voltages, stimulated by flufenamic acid, blocked by Ba2+, and insensitive to diltiazem; an inwardly rectifying K+ current; and a nonselective cation current inhibited by flufenamic acid. CONCLUSIONS. Our results are consistent with those in previous studies of noncultured epithelia from rabbit and human corneas showing an outwardly rectifying K+ current active at hyperpolarized voltages and a nonselective cation current active at depolarized voltages and insensitive to Ba2+. These data suggest cultured cells may be useful in determining the physiological role of ion channels in corneal epithelia and may aid in the development of a cell-based model for the examination of the effects of wounding and toxic agents on the human cornea.

Original languageEnglish (US)
Pages (from-to)1143-1151
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume39
Issue number7
StatePublished - Jun 1 1998
Externally publishedYes

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Epithelial Cells
Flufenamic Acid
Cornea
Cations
Corneal Epithelium
Cultured Cells
Tetraethylammonium
Diltiazem
Poisons
Amphotericin B
Keratins
Ion Channels
Epithelium
Rabbits

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Properties of whole-cell ionic currents in cultured human corneal epithelial cells. / Bockman, Charles S.; Griffith, May; Watsky, Mitchell Aaron.

In: Investigative Ophthalmology and Visual Science, Vol. 39, No. 7, 01.06.1998, p. 1143-1151.

Research output: Contribution to journalArticle

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N2 - PURPOSE. To identify and partially characterize the ionic currents contributing to the whole-cell conductance of cultured human corneal epithelial cells. METHODS. Epithelial cells were scraped from human donor corneas and cultured for use in patch-clamp experiments. Amphotericin B and the perforated-patch configuration were used to measure whole-cell currents in cells isolated from confluent monolayers. RESULTS. Cell monolayers exhibited cobblestone morphology and were immunopositive for corneal epithelium-specific cytokeratin. Single cells had a capacitance of 21 ≠ 2 pF and expressed similar types of ionic currents regardless of passage number. In descending order of frequency of occurrence, cells exhibited a nonselective cation current active at depolarized voltages and insensitive to Ba2+ and Gd3+; an outwardly rectifying K+ current active at depolarized voltages, stimulated by flufenamic acid and inhibited by tetraethylammonium; a voltage-gated inward Na+ current; an outwardly rectifying K+ current active at hyperpolarized voltages, stimulated by flufenamic acid, blocked by Ba2+, and insensitive to diltiazem; an inwardly rectifying K+ current; and a nonselective cation current inhibited by flufenamic acid. CONCLUSIONS. Our results are consistent with those in previous studies of noncultured epithelia from rabbit and human corneas showing an outwardly rectifying K+ current active at hyperpolarized voltages and a nonselective cation current active at depolarized voltages and insensitive to Ba2+. These data suggest cultured cells may be useful in determining the physiological role of ion channels in corneal epithelia and may aid in the development of a cell-based model for the examination of the effects of wounding and toxic agents on the human cornea.

AB - PURPOSE. To identify and partially characterize the ionic currents contributing to the whole-cell conductance of cultured human corneal epithelial cells. METHODS. Epithelial cells were scraped from human donor corneas and cultured for use in patch-clamp experiments. Amphotericin B and the perforated-patch configuration were used to measure whole-cell currents in cells isolated from confluent monolayers. RESULTS. Cell monolayers exhibited cobblestone morphology and were immunopositive for corneal epithelium-specific cytokeratin. Single cells had a capacitance of 21 ≠ 2 pF and expressed similar types of ionic currents regardless of passage number. In descending order of frequency of occurrence, cells exhibited a nonselective cation current active at depolarized voltages and insensitive to Ba2+ and Gd3+; an outwardly rectifying K+ current active at depolarized voltages, stimulated by flufenamic acid and inhibited by tetraethylammonium; a voltage-gated inward Na+ current; an outwardly rectifying K+ current active at hyperpolarized voltages, stimulated by flufenamic acid, blocked by Ba2+, and insensitive to diltiazem; an inwardly rectifying K+ current; and a nonselective cation current inhibited by flufenamic acid. CONCLUSIONS. Our results are consistent with those in previous studies of noncultured epithelia from rabbit and human corneas showing an outwardly rectifying K+ current active at hyperpolarized voltages and a nonselective cation current active at depolarized voltages and insensitive to Ba2+. These data suggest cultured cells may be useful in determining the physiological role of ion channels in corneal epithelia and may aid in the development of a cell-based model for the examination of the effects of wounding and toxic agents on the human cornea.

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