Store-operated Ca2+ channels in human glomerular mesangial cells

Rong Ma, Sonja Smith, Angie Child, Pamela K Carmines, Steven Claude Sansom

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

Experiments were performed to identify the biophysical properties of store-operated Ca2+ channels (SOC) in cultured human glomerular mesangial cells (MC). A fluorometric technique (fura 2) was utilized to monitor the change in intracellular calcium concentration ([Ca2+](i)) evoked by elevating external [Ca2+] from 10 nM to 1 mM (Δ[Ca2+]). Under control conditions, Δ[Ca2+] averaged 6 nM and was unaffected by elevating bath [K+]. After treatment with 1 μM thapsigargin to deplete the intracellular Ca2+ store, the change in [Ca2+](i) (Δ[Ca2+](th)) averaged 147 ± 16 nM. In thapsigargin-treated MC Studied under depolarizing conditions (75 mM bath K+), Δ[Ca2+](th) was 45 ± 7 nM. The Δ[Ca2+](th) response of thapsigargin-treated cells was inhibited by La3+ (IC50 = 335 nM) but was unaffected by 5 μM Cd2+. In patch clamp studies, inward currents were observed in cell-attached patches with either 90 mM Ba2+ or Ca2+ in the pipette and 140 mM KCl in the bathing solution. The single-channel conductance was 2.1 pS with Ba2+ and 0.7 pS with Ca2+. The estimated selectivities were Ca2+ > Ba2+ >> K+. These channels were sensitive to 2 μM La3+, insensitive to 5 μM Cd2+, and voltage independent, with an average channel activity (NP0) of 1.02 at command potential (-V(p)) ranging from 0 to -80 mV. In summary, MC exhibited an electrogenic Ca2+ influx pathway that is suggestive of Ca2+ entry through SOC, as well as a small- conductance divalent-selective channel displaying biophysical properties consistent with SOC. Based on estimates of whole cell Ca2+ influx derived from our data, we conclude that SOC with low single-channel conductance must be highly abundant in MC to allow significant capacitative Ca2+ entry in response to depletion of the intracellular store.

Original languageEnglish (US)
Pages (from-to)F954-F961
JournalAmerican Journal of Physiology - Renal Physiology
Volume278
Issue number6 47-6
StatePublished - Jun 1 2000

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Mesangial Cells
Thapsigargin
Baths
Inhibitory Concentration 50
Calcium
Therapeutics

Keywords

  • Angiotensin II
  • Cadmium
  • Fura 2 fluorescence
  • Lanthanum
  • Patch clamp
  • Thapsigargin

ASJC Scopus subject areas

  • Physiology
  • Urology

Cite this

Store-operated Ca2+ channels in human glomerular mesangial cells. / Ma, Rong; Smith, Sonja; Child, Angie; Carmines, Pamela K; Sansom, Steven Claude.

In: American Journal of Physiology - Renal Physiology, Vol. 278, No. 6 47-6, 01.06.2000, p. F954-F961.

Research output: Contribution to journalArticle

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abstract = "Experiments were performed to identify the biophysical properties of store-operated Ca2+ channels (SOC) in cultured human glomerular mesangial cells (MC). A fluorometric technique (fura 2) was utilized to monitor the change in intracellular calcium concentration ([Ca2+](i)) evoked by elevating external [Ca2+] from 10 nM to 1 mM (Δ[Ca2+]). Under control conditions, Δ[Ca2+] averaged 6 nM and was unaffected by elevating bath [K+]. After treatment with 1 μM thapsigargin to deplete the intracellular Ca2+ store, the change in [Ca2+](i) (Δ[Ca2+](th)) averaged 147 ± 16 nM. In thapsigargin-treated MC Studied under depolarizing conditions (75 mM bath K+), Δ[Ca2+](th) was 45 ± 7 nM. The Δ[Ca2+](th) response of thapsigargin-treated cells was inhibited by La3+ (IC50 = 335 nM) but was unaffected by 5 μM Cd2+. In patch clamp studies, inward currents were observed in cell-attached patches with either 90 mM Ba2+ or Ca2+ in the pipette and 140 mM KCl in the bathing solution. The single-channel conductance was 2.1 pS with Ba2+ and 0.7 pS with Ca2+. The estimated selectivities were Ca2+ > Ba2+ >> K+. These channels were sensitive to 2 μM La3+, insensitive to 5 μM Cd2+, and voltage independent, with an average channel activity (NP0) of 1.02 at command potential (-V(p)) ranging from 0 to -80 mV. In summary, MC exhibited an electrogenic Ca2+ influx pathway that is suggestive of Ca2+ entry through SOC, as well as a small- conductance divalent-selective channel displaying biophysical properties consistent with SOC. Based on estimates of whole cell Ca2+ influx derived from our data, we conclude that SOC with low single-channel conductance must be highly abundant in MC to allow significant capacitative Ca2+ entry in response to depletion of the intracellular store.",
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T1 - Store-operated Ca2+ channels in human glomerular mesangial cells

AU - Ma, Rong

AU - Smith, Sonja

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AU - Carmines, Pamela K

AU - Sansom, Steven Claude

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N2 - Experiments were performed to identify the biophysical properties of store-operated Ca2+ channels (SOC) in cultured human glomerular mesangial cells (MC). A fluorometric technique (fura 2) was utilized to monitor the change in intracellular calcium concentration ([Ca2+](i)) evoked by elevating external [Ca2+] from 10 nM to 1 mM (Δ[Ca2+]). Under control conditions, Δ[Ca2+] averaged 6 nM and was unaffected by elevating bath [K+]. After treatment with 1 μM thapsigargin to deplete the intracellular Ca2+ store, the change in [Ca2+](i) (Δ[Ca2+](th)) averaged 147 ± 16 nM. In thapsigargin-treated MC Studied under depolarizing conditions (75 mM bath K+), Δ[Ca2+](th) was 45 ± 7 nM. The Δ[Ca2+](th) response of thapsigargin-treated cells was inhibited by La3+ (IC50 = 335 nM) but was unaffected by 5 μM Cd2+. In patch clamp studies, inward currents were observed in cell-attached patches with either 90 mM Ba2+ or Ca2+ in the pipette and 140 mM KCl in the bathing solution. The single-channel conductance was 2.1 pS with Ba2+ and 0.7 pS with Ca2+. The estimated selectivities were Ca2+ > Ba2+ >> K+. These channels were sensitive to 2 μM La3+, insensitive to 5 μM Cd2+, and voltage independent, with an average channel activity (NP0) of 1.02 at command potential (-V(p)) ranging from 0 to -80 mV. In summary, MC exhibited an electrogenic Ca2+ influx pathway that is suggestive of Ca2+ entry through SOC, as well as a small- conductance divalent-selective channel displaying biophysical properties consistent with SOC. Based on estimates of whole cell Ca2+ influx derived from our data, we conclude that SOC with low single-channel conductance must be highly abundant in MC to allow significant capacitative Ca2+ entry in response to depletion of the intracellular store.

AB - Experiments were performed to identify the biophysical properties of store-operated Ca2+ channels (SOC) in cultured human glomerular mesangial cells (MC). A fluorometric technique (fura 2) was utilized to monitor the change in intracellular calcium concentration ([Ca2+](i)) evoked by elevating external [Ca2+] from 10 nM to 1 mM (Δ[Ca2+]). Under control conditions, Δ[Ca2+] averaged 6 nM and was unaffected by elevating bath [K+]. After treatment with 1 μM thapsigargin to deplete the intracellular Ca2+ store, the change in [Ca2+](i) (Δ[Ca2+](th)) averaged 147 ± 16 nM. In thapsigargin-treated MC Studied under depolarizing conditions (75 mM bath K+), Δ[Ca2+](th) was 45 ± 7 nM. The Δ[Ca2+](th) response of thapsigargin-treated cells was inhibited by La3+ (IC50 = 335 nM) but was unaffected by 5 μM Cd2+. In patch clamp studies, inward currents were observed in cell-attached patches with either 90 mM Ba2+ or Ca2+ in the pipette and 140 mM KCl in the bathing solution. The single-channel conductance was 2.1 pS with Ba2+ and 0.7 pS with Ca2+. The estimated selectivities were Ca2+ > Ba2+ >> K+. These channels were sensitive to 2 μM La3+, insensitive to 5 μM Cd2+, and voltage independent, with an average channel activity (NP0) of 1.02 at command potential (-V(p)) ranging from 0 to -80 mV. In summary, MC exhibited an electrogenic Ca2+ influx pathway that is suggestive of Ca2+ entry through SOC, as well as a small- conductance divalent-selective channel displaying biophysical properties consistent with SOC. Based on estimates of whole cell Ca2+ influx derived from our data, we conclude that SOC with low single-channel conductance must be highly abundant in MC to allow significant capacitative Ca2+ entry in response to depletion of the intracellular store.

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