Maitotoxin activates an endogenous non-selective cation channel and is an effective initiator of the activation of the heterologously expressed hTRPC-1 (transient receptor potential) non-selective cation channel in H4-IIE liver cells

Helen M. Brereton, Jinglong Chen, Grigori Rychkov, M. Lyn Harland, Gregory J. Barritt

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Abstract

The structures and mechanisms of activation of non-selective cation channels (NSCCs) are not well understood although NSCCs play important roles in the regulation of metabolism, ion transport, cell volume and cell shape. It has been proposed that TRP (transient receptor potential) proteins are the molecular correlates of some NSCCs. Using fura-2 and patch-clamp recording, it was shown that the maitotoxin-activated cation channels in the H4-IIE rat liver cell line admit Ca2+, Mn2+ and Na+, have a high selectivity for Na+ compared with Ca2+, and are inhibited by Gd3+ (half-maximal inhibition at 1 μM). Activation of the channels by maitotoxin was inhibited by increasing the extracellular Ca2+ concentration or by inclusion of 10 mM EGTA in the patch pipette. mRNA encoding TRP proteins 1, 2 and 3 at levels comparable with those in brain was detected using reverse transcriptase-polymerase chain reaction in poly(A)+ RNA prepared from H4-IIE cells and freshly-isolated rat hepatocytes. In H4-IIE cells transiently transfected with cDNA encoding hTRPC-1, the expressed hTRPC-1 protein was chiefly located at intracellular sites and at the plasma membrane. Cells expressing hTRPC-1 exhibited a substantial enhancement of maitotoxin-initiated Ca2+ inflow and a modest enhancement of thapsigargin-initiated Ca2+ inflow (measured using fura-2) and no enhancement of the highly Ca2+-selective store-operated Ca2+ current (measured using patch-clamp recording). In cells expressing hTRPC-1, maitotoxin activated channels which were not found in untransfected cells, have an approximately equal selectivity for Na+ and Ca2+, and are inhibited by Gd3+ (half-maximal inhibition at 3 μM). It is concluded that in liver cells (i) maitotoxin initiates the activation of endogenous NSCCs with a high selectivity for Na+ compared with Ca2+; (ii) TRP proteins 1, 2 and 3 are expressed; (iii) maitotoxin is an effective initiator of activation of heterologously expressed hTRPC-1 channels; and (iv) the endogenous TRP-1 protein is unlikely to be the molecular counterpart of the maitotoxin-activated NSCCs nor the highly Ca2+-selective store-operated Ca2+ channels.

LanguageEnglish
Pages107-126
Number of pages20
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1540
Issue number2
DOIs
Publication statusPublished - 22 Aug 2001

Keywords

  • Ca, Mn and Na inflow
  • Gd
  • H4-IIE cell
  • Heterologous expression
  • Patch-clamp recording
  • Reverse transcriptase-polymerase chain reaction

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

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title = "Maitotoxin activates an endogenous non-selective cation channel and is an effective initiator of the activation of the heterologously expressed hTRPC-1 (transient receptor potential) non-selective cation channel in H4-IIE liver cells",
abstract = "The structures and mechanisms of activation of non-selective cation channels (NSCCs) are not well understood although NSCCs play important roles in the regulation of metabolism, ion transport, cell volume and cell shape. It has been proposed that TRP (transient receptor potential) proteins are the molecular correlates of some NSCCs. Using fura-2 and patch-clamp recording, it was shown that the maitotoxin-activated cation channels in the H4-IIE rat liver cell line admit Ca2+, Mn2+ and Na+, have a high selectivity for Na+ compared with Ca2+, and are inhibited by Gd3+ (half-maximal inhibition at 1 μM). Activation of the channels by maitotoxin was inhibited by increasing the extracellular Ca2+ concentration or by inclusion of 10 mM EGTA in the patch pipette. mRNA encoding TRP proteins 1, 2 and 3 at levels comparable with those in brain was detected using reverse transcriptase-polymerase chain reaction in poly(A)+ RNA prepared from H4-IIE cells and freshly-isolated rat hepatocytes. In H4-IIE cells transiently transfected with cDNA encoding hTRPC-1, the expressed hTRPC-1 protein was chiefly located at intracellular sites and at the plasma membrane. Cells expressing hTRPC-1 exhibited a substantial enhancement of maitotoxin-initiated Ca2+ inflow and a modest enhancement of thapsigargin-initiated Ca2+ inflow (measured using fura-2) and no enhancement of the highly Ca2+-selective store-operated Ca2+ current (measured using patch-clamp recording). In cells expressing hTRPC-1, maitotoxin activated channels which were not found in untransfected cells, have an approximately equal selectivity for Na+ and Ca2+, and are inhibited by Gd3+ (half-maximal inhibition at 3 μM). It is concluded that in liver cells (i) maitotoxin initiates the activation of endogenous NSCCs with a high selectivity for Na+ compared with Ca2+; (ii) TRP proteins 1, 2 and 3 are expressed; (iii) maitotoxin is an effective initiator of activation of heterologously expressed hTRPC-1 channels; and (iv) the endogenous TRP-1 protein is unlikely to be the molecular counterpart of the maitotoxin-activated NSCCs nor the highly Ca2+-selective store-operated Ca2+ channels.",
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author = "Brereton, {Helen M.} and Jinglong Chen and Grigori Rychkov and Harland, {M. Lyn} and Barritt, {Gregory J.}",
year = "2001",
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T1 - Maitotoxin activates an endogenous non-selective cation channel and is an effective initiator of the activation of the heterologously expressed hTRPC-1 (transient receptor potential) non-selective cation channel in H4-IIE liver cells

AU - Brereton, Helen M.

AU - Chen, Jinglong

AU - Rychkov, Grigori

AU - Harland, M. Lyn

AU - Barritt, Gregory J.

PY - 2001/8/22

Y1 - 2001/8/22

N2 - The structures and mechanisms of activation of non-selective cation channels (NSCCs) are not well understood although NSCCs play important roles in the regulation of metabolism, ion transport, cell volume and cell shape. It has been proposed that TRP (transient receptor potential) proteins are the molecular correlates of some NSCCs. Using fura-2 and patch-clamp recording, it was shown that the maitotoxin-activated cation channels in the H4-IIE rat liver cell line admit Ca2+, Mn2+ and Na+, have a high selectivity for Na+ compared with Ca2+, and are inhibited by Gd3+ (half-maximal inhibition at 1 μM). Activation of the channels by maitotoxin was inhibited by increasing the extracellular Ca2+ concentration or by inclusion of 10 mM EGTA in the patch pipette. mRNA encoding TRP proteins 1, 2 and 3 at levels comparable with those in brain was detected using reverse transcriptase-polymerase chain reaction in poly(A)+ RNA prepared from H4-IIE cells and freshly-isolated rat hepatocytes. In H4-IIE cells transiently transfected with cDNA encoding hTRPC-1, the expressed hTRPC-1 protein was chiefly located at intracellular sites and at the plasma membrane. Cells expressing hTRPC-1 exhibited a substantial enhancement of maitotoxin-initiated Ca2+ inflow and a modest enhancement of thapsigargin-initiated Ca2+ inflow (measured using fura-2) and no enhancement of the highly Ca2+-selective store-operated Ca2+ current (measured using patch-clamp recording). In cells expressing hTRPC-1, maitotoxin activated channels which were not found in untransfected cells, have an approximately equal selectivity for Na+ and Ca2+, and are inhibited by Gd3+ (half-maximal inhibition at 3 μM). It is concluded that in liver cells (i) maitotoxin initiates the activation of endogenous NSCCs with a high selectivity for Na+ compared with Ca2+; (ii) TRP proteins 1, 2 and 3 are expressed; (iii) maitotoxin is an effective initiator of activation of heterologously expressed hTRPC-1 channels; and (iv) the endogenous TRP-1 protein is unlikely to be the molecular counterpart of the maitotoxin-activated NSCCs nor the highly Ca2+-selective store-operated Ca2+ channels.

AB - The structures and mechanisms of activation of non-selective cation channels (NSCCs) are not well understood although NSCCs play important roles in the regulation of metabolism, ion transport, cell volume and cell shape. It has been proposed that TRP (transient receptor potential) proteins are the molecular correlates of some NSCCs. Using fura-2 and patch-clamp recording, it was shown that the maitotoxin-activated cation channels in the H4-IIE rat liver cell line admit Ca2+, Mn2+ and Na+, have a high selectivity for Na+ compared with Ca2+, and are inhibited by Gd3+ (half-maximal inhibition at 1 μM). Activation of the channels by maitotoxin was inhibited by increasing the extracellular Ca2+ concentration or by inclusion of 10 mM EGTA in the patch pipette. mRNA encoding TRP proteins 1, 2 and 3 at levels comparable with those in brain was detected using reverse transcriptase-polymerase chain reaction in poly(A)+ RNA prepared from H4-IIE cells and freshly-isolated rat hepatocytes. In H4-IIE cells transiently transfected with cDNA encoding hTRPC-1, the expressed hTRPC-1 protein was chiefly located at intracellular sites and at the plasma membrane. Cells expressing hTRPC-1 exhibited a substantial enhancement of maitotoxin-initiated Ca2+ inflow and a modest enhancement of thapsigargin-initiated Ca2+ inflow (measured using fura-2) and no enhancement of the highly Ca2+-selective store-operated Ca2+ current (measured using patch-clamp recording). In cells expressing hTRPC-1, maitotoxin activated channels which were not found in untransfected cells, have an approximately equal selectivity for Na+ and Ca2+, and are inhibited by Gd3+ (half-maximal inhibition at 3 μM). It is concluded that in liver cells (i) maitotoxin initiates the activation of endogenous NSCCs with a high selectivity for Na+ compared with Ca2+; (ii) TRP proteins 1, 2 and 3 are expressed; (iii) maitotoxin is an effective initiator of activation of heterologously expressed hTRPC-1 channels; and (iv) the endogenous TRP-1 protein is unlikely to be the molecular counterpart of the maitotoxin-activated NSCCs nor the highly Ca2+-selective store-operated Ca2+ channels.

KW - Ca, Mn and Na inflow

KW - Gd

KW - H4-IIE cell

KW - Heterologous expression

KW - Patch-clamp recording

KW - Reverse transcriptase-polymerase chain reaction

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U2 - 10.1016/S0167-4889(01)00124-0

DO - 10.1016/S0167-4889(01)00124-0

M3 - Article

VL - 1540

SP - 107

EP - 126

JO - Biochimica et Biophysica Acta - Molecular Cell Research

T2 - Biochimica et Biophysica Acta - Molecular Cell Research

JF - Biochimica et Biophysica Acta - Molecular Cell Research

SN - 0167-4889

IS - 2

ER -