The effects of trypsin on ATP-sensitive potassium channel properties and sulfonylurea receptors in the CRI-G1 insulin-secreting cell line

K. Lee, S. E. Ozanne, I. C. M. Rowe, C. N. Hales, M. L. J. Ashford

    Research output: Contribution to journalArticle

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    Abstract

    The effects of the proteolytic enzyme trypsin upon ATP-sensitive potassium (KATP) channel activity were examined in the CRI-G1 insulin-secreting cell line. Trypsin activated channels only when applied to the intracellular surface of the cell membrane. The activation could be prevented by the concomitant application of trypsin inhibitor or by heat inactivation of the enzyme. The trypsin-induced change in channel activity was accompanied by a reduction in the rate of channel rundown. However, trypsin did not affect the mean single channel conductance (55.2 pS), the ionic selectivity, or rectification of the KATP channel. Concentration response curves for various KATP channel inhibitors were constructed in the presence and absence of intracellular trypsin. The EC50 for tolbutamide was shifted from 30.0 +/- 4.5 microM, with 100 micrograms/ml heat-inactivated trypsin present to 9.7 +/- 1.0 mM with active trypsin in the intracellular solution. Treatment of the cells' external surface with 1 mg/ml trypsin did not alter the potency of tolbutamide. Intracellular trypsin also produced a significant fall in the potency of glibenclamide, meglitinide, and phentolamine but did not alter the effectiveness of thiopentone. Radioligand binding studies demonstrated a total loss of 3H-labeled glibenclamide binding when the intracellular surface of the cells was exposed to trypsin. In contrast, 3H-labeled glibenclamide binding was not affected when the enzyme was applied to the external surface. Trypsin treatment, therefore, alters a number of characteristics of KATP channel pharmacology, and we suggest that this is due to action at possibly more than one site but includes the functional cleavage of the sulfonylurea receptor from the KATP channel.
    Original languageEnglish
    Pages (from-to)176-185
    Number of pages10
    JournalMolecular Pharmacology
    Volume46
    Issue number1
    Publication statusPublished - Jul 1994

    Fingerprint

    Sulfonylurea Receptors
    KATP Channels
    Insulin-Secreting Cells
    Trypsin
    Cell Line
    Glyburide
    Tolbutamide
    Hot Temperature
    Trypsin Inhibitors
    Thiopental
    Phentolamine
    Enzymes

    Keywords

    • Glyburide
    • Animals
    • Trypsin
    • Islets of Langerhans
    • Insulin
    • Potassium Channels
    • Rats
    • Receptors, Drug
    • Potassium Channels, Inwardly Rectifying
    • ATP-Binding Cassette Transporters
    • Membrane Potentials
    • Adenosine Triphosphate
    • Tolbutamide
    • Cell Line

    Cite this

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    title = "The effects of trypsin on ATP-sensitive potassium channel properties and sulfonylurea receptors in the CRI-G1 insulin-secreting cell line",
    abstract = "The effects of the proteolytic enzyme trypsin upon ATP-sensitive potassium (KATP) channel activity were examined in the CRI-G1 insulin-secreting cell line. Trypsin activated channels only when applied to the intracellular surface of the cell membrane. The activation could be prevented by the concomitant application of trypsin inhibitor or by heat inactivation of the enzyme. The trypsin-induced change in channel activity was accompanied by a reduction in the rate of channel rundown. However, trypsin did not affect the mean single channel conductance (55.2 pS), the ionic selectivity, or rectification of the KATP channel. Concentration response curves for various KATP channel inhibitors were constructed in the presence and absence of intracellular trypsin. The EC50 for tolbutamide was shifted from 30.0 +/- 4.5 microM, with 100 micrograms/ml heat-inactivated trypsin present to 9.7 +/- 1.0 mM with active trypsin in the intracellular solution. Treatment of the cells' external surface with 1 mg/ml trypsin did not alter the potency of tolbutamide. Intracellular trypsin also produced a significant fall in the potency of glibenclamide, meglitinide, and phentolamine but did not alter the effectiveness of thiopentone. Radioligand binding studies demonstrated a total loss of 3H-labeled glibenclamide binding when the intracellular surface of the cells was exposed to trypsin. In contrast, 3H-labeled glibenclamide binding was not affected when the enzyme was applied to the external surface. Trypsin treatment, therefore, alters a number of characteristics of KATP channel pharmacology, and we suggest that this is due to action at possibly more than one site but includes the functional cleavage of the sulfonylurea receptor from the KATP channel.",
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    author = "K. Lee and Ozanne, {S. E.} and Rowe, {I. C. M.} and Hales, {C. N.} and Ashford, {M. L. J.}",
    year = "1994",
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    pages = "176--185",
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    The effects of trypsin on ATP-sensitive potassium channel properties and sulfonylurea receptors in the CRI-G1 insulin-secreting cell line. / Lee, K.; Ozanne, S. E.; Rowe, I. C. M.; Hales, C. N.; Ashford, M. L. J.

    In: Molecular Pharmacology, Vol. 46, No. 1, 07.1994, p. 176-185.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - The effects of trypsin on ATP-sensitive potassium channel properties and sulfonylurea receptors in the CRI-G1 insulin-secreting cell line

    AU - Lee, K.

    AU - Ozanne, S. E.

    AU - Rowe, I. C. M.

    AU - Hales, C. N.

    AU - Ashford, M. L. J.

    PY - 1994/7

    Y1 - 1994/7

    N2 - The effects of the proteolytic enzyme trypsin upon ATP-sensitive potassium (KATP) channel activity were examined in the CRI-G1 insulin-secreting cell line. Trypsin activated channels only when applied to the intracellular surface of the cell membrane. The activation could be prevented by the concomitant application of trypsin inhibitor or by heat inactivation of the enzyme. The trypsin-induced change in channel activity was accompanied by a reduction in the rate of channel rundown. However, trypsin did not affect the mean single channel conductance (55.2 pS), the ionic selectivity, or rectification of the KATP channel. Concentration response curves for various KATP channel inhibitors were constructed in the presence and absence of intracellular trypsin. The EC50 for tolbutamide was shifted from 30.0 +/- 4.5 microM, with 100 micrograms/ml heat-inactivated trypsin present to 9.7 +/- 1.0 mM with active trypsin in the intracellular solution. Treatment of the cells' external surface with 1 mg/ml trypsin did not alter the potency of tolbutamide. Intracellular trypsin also produced a significant fall in the potency of glibenclamide, meglitinide, and phentolamine but did not alter the effectiveness of thiopentone. Radioligand binding studies demonstrated a total loss of 3H-labeled glibenclamide binding when the intracellular surface of the cells was exposed to trypsin. In contrast, 3H-labeled glibenclamide binding was not affected when the enzyme was applied to the external surface. Trypsin treatment, therefore, alters a number of characteristics of KATP channel pharmacology, and we suggest that this is due to action at possibly more than one site but includes the functional cleavage of the sulfonylurea receptor from the KATP channel.

    AB - The effects of the proteolytic enzyme trypsin upon ATP-sensitive potassium (KATP) channel activity were examined in the CRI-G1 insulin-secreting cell line. Trypsin activated channels only when applied to the intracellular surface of the cell membrane. The activation could be prevented by the concomitant application of trypsin inhibitor or by heat inactivation of the enzyme. The trypsin-induced change in channel activity was accompanied by a reduction in the rate of channel rundown. However, trypsin did not affect the mean single channel conductance (55.2 pS), the ionic selectivity, or rectification of the KATP channel. Concentration response curves for various KATP channel inhibitors were constructed in the presence and absence of intracellular trypsin. The EC50 for tolbutamide was shifted from 30.0 +/- 4.5 microM, with 100 micrograms/ml heat-inactivated trypsin present to 9.7 +/- 1.0 mM with active trypsin in the intracellular solution. Treatment of the cells' external surface with 1 mg/ml trypsin did not alter the potency of tolbutamide. Intracellular trypsin also produced a significant fall in the potency of glibenclamide, meglitinide, and phentolamine but did not alter the effectiveness of thiopentone. Radioligand binding studies demonstrated a total loss of 3H-labeled glibenclamide binding when the intracellular surface of the cells was exposed to trypsin. In contrast, 3H-labeled glibenclamide binding was not affected when the enzyme was applied to the external surface. Trypsin treatment, therefore, alters a number of characteristics of KATP channel pharmacology, and we suggest that this is due to action at possibly more than one site but includes the functional cleavage of the sulfonylurea receptor from the KATP channel.

    KW - Glyburide

    KW - Animals

    KW - Trypsin

    KW - Islets of Langerhans

    KW - Insulin

    KW - Potassium Channels

    KW - Rats

    KW - Receptors, Drug

    KW - Potassium Channels, Inwardly Rectifying

    KW - ATP-Binding Cassette Transporters

    KW - Membrane Potentials

    KW - Adenosine Triphosphate

    KW - Tolbutamide

    KW - Cell Line

    M3 - Article

    VL - 46

    SP - 176

    EP - 185

    JO - Molecular Pharmacology

    JF - Molecular Pharmacology

    SN - 0026-895X

    IS - 1

    ER -