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Functional ryanodine receptors in the plasma membrane of RINm5F pancreatic beta-cells.

Rosker C, Meur G, Taylor EJ, Taylor CW - J. Biol. Chem. (2008)

Bottom Line: Subcellular fractionation showed that type 3 IP(3) receptors (IP(3)R3) were expressed predominantly in endoplasmic reticulum, whereas RyR2 were present also in plasma membrane fractions.Using RNAi selectively to reduce expression of RyR1, RyR2, or IP(3)R3, we showed that RyR2 mediates both the Ca(2+) entry and the plasma membrane currents evoked by agonists of RyR.We conclude that small numbers of RyR2 are selectively expressed in the plasma membrane of RINm5F pancreatic beta-cells, where they mediate Ca(2+) entry.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom.

ABSTRACT
Ryanodine receptors (RyR) are Ca(2+) channels that mediate Ca(2+) release from intracellular stores in response to diverse intracellular signals. In RINm5F insulinoma cells, caffeine, and 4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca(2+) entry that was independent of store-operated Ca(2+) entry, and blocked by prior incubation with a concentration of ryanodine that inactivates RyR. Patch-clamp recording identified small numbers of large-conductance (gamma(K) = 169 pS) cation channels that were activated by caffeine, 4CmC or low concentrations of ryanodine. Similar channels were detected in rat pancreatic beta-cells. In RINm5F cells, the channels were blocked by cytosolic, but not extracellular, ruthenium red. Subcellular fractionation showed that type 3 IP(3) receptors (IP(3)R3) were expressed predominantly in endoplasmic reticulum, whereas RyR2 were present also in plasma membrane fractions. Using RNAi selectively to reduce expression of RyR1, RyR2, or IP(3)R3, we showed that RyR2 mediates both the Ca(2+) entry and the plasma membrane currents evoked by agonists of RyR. We conclude that small numbers of RyR2 are selectively expressed in the plasma membrane of RINm5F pancreatic beta-cells, where they mediate Ca(2+) entry.

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Ca2+ entry evoked by agonists of RyR. A, Ca2+ signals evoked by 4CmC (1 mm) in HBS with or without extracellular Ca2+ and after pretreatment with ryanodine (400 μm, 30 min). Traces (here and in subsequent panels) show the responses from ∼50 individual cells taken from at least three independent experiments (means ± S.E., but with many error bars smaller than the symbols). B, responses to caffeine (1 mm) in normal HBS. C, Ca2+ signals evoked by 4CmC (1 mm) in Ca2+-free medium alone or after pretreatment with thapsigargin (TG, 1 μm). D, peak (after ∼30 s) and sustained (after 200 s) changes in [Ca2+]i evoked by 4CmC in the absence (open bars) and presence of extracellular Ca2+, and the latter after pretreatment (2 min) with nifedipine (Nif, 10 μm) or thapsigargin (1 μm). E, Mn2+ entry (0.5 mm) was measured by quenching of Fura 2 fluorescence in control cells (Q1). Mn2+ was then removed and the cells incubated with thapsigargin (1 μm) for 5 min (during the break between the traces) before again measuring Mn2+ entry (Q2) in the presence of thapsigargin alone (black) or with 4CmC (1 mm, red). Typical traces are shown. Summary data show the slope of the fluorescence change during the second addition of Mn2+ (Q2, with the stimulus) as a % (Q2/Q1) of the slope recorded during the first addition (Q1, without stimulus). Results are means ± S.E., n = 30–61. F, restoration of extracellular Ca2+ (1.5 mm) to cells pretreated with thapsigargin (1 μm) in Ca2+-free HBS fails to evoke Ca2+ entry.
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fig1: Ca2+ entry evoked by agonists of RyR. A, Ca2+ signals evoked by 4CmC (1 mm) in HBS with or without extracellular Ca2+ and after pretreatment with ryanodine (400 μm, 30 min). Traces (here and in subsequent panels) show the responses from ∼50 individual cells taken from at least three independent experiments (means ± S.E., but with many error bars smaller than the symbols). B, responses to caffeine (1 mm) in normal HBS. C, Ca2+ signals evoked by 4CmC (1 mm) in Ca2+-free medium alone or after pretreatment with thapsigargin (TG, 1 μm). D, peak (after ∼30 s) and sustained (after 200 s) changes in [Ca2+]i evoked by 4CmC in the absence (open bars) and presence of extracellular Ca2+, and the latter after pretreatment (2 min) with nifedipine (Nif, 10 μm) or thapsigargin (1 μm). E, Mn2+ entry (0.5 mm) was measured by quenching of Fura 2 fluorescence in control cells (Q1). Mn2+ was then removed and the cells incubated with thapsigargin (1 μm) for 5 min (during the break between the traces) before again measuring Mn2+ entry (Q2) in the presence of thapsigargin alone (black) or with 4CmC (1 mm, red). Typical traces are shown. Summary data show the slope of the fluorescence change during the second addition of Mn2+ (Q2, with the stimulus) as a % (Q2/Q1) of the slope recorded during the first addition (Q1, without stimulus). Results are means ± S.E., n = 30–61. F, restoration of extracellular Ca2+ (1.5 mm) to cells pretreated with thapsigargin (1 μm) in Ca2+-free HBS fails to evoke Ca2+ entry.

Mentions: Quenching of fura 2 fluorescence by Mn2+ entry was measured in similarly loaded cells bathed in Ca2+-free HBS supplemented with 0.5 mm MnCl2 and with fluorescence recorded after excitation at 360 nm. For all experiments, a basal rate of quench (Q1) was measured before stimulation, and the rate was again measured after stimulation (Q2). The duration of the exposure to MnCl2 (100 s) was chosen to ensure that rates of Fura 2 quenching were linear during both assay periods. The effects of stimuli on rates of Mn2+ entry are reported as a Q2/Q1 ratio, with the ratio uniquely determined for each single cell (see Fig. 1E).


Functional ryanodine receptors in the plasma membrane of RINm5F pancreatic beta-cells.

Rosker C, Meur G, Taylor EJ, Taylor CW - J. Biol. Chem. (2008)

Ca2+ entry evoked by agonists of RyR. A, Ca2+ signals evoked by 4CmC (1 mm) in HBS with or without extracellular Ca2+ and after pretreatment with ryanodine (400 μm, 30 min). Traces (here and in subsequent panels) show the responses from ∼50 individual cells taken from at least three independent experiments (means ± S.E., but with many error bars smaller than the symbols). B, responses to caffeine (1 mm) in normal HBS. C, Ca2+ signals evoked by 4CmC (1 mm) in Ca2+-free medium alone or after pretreatment with thapsigargin (TG, 1 μm). D, peak (after ∼30 s) and sustained (after 200 s) changes in [Ca2+]i evoked by 4CmC in the absence (open bars) and presence of extracellular Ca2+, and the latter after pretreatment (2 min) with nifedipine (Nif, 10 μm) or thapsigargin (1 μm). E, Mn2+ entry (0.5 mm) was measured by quenching of Fura 2 fluorescence in control cells (Q1). Mn2+ was then removed and the cells incubated with thapsigargin (1 μm) for 5 min (during the break between the traces) before again measuring Mn2+ entry (Q2) in the presence of thapsigargin alone (black) or with 4CmC (1 mm, red). Typical traces are shown. Summary data show the slope of the fluorescence change during the second addition of Mn2+ (Q2, with the stimulus) as a % (Q2/Q1) of the slope recorded during the first addition (Q1, without stimulus). Results are means ± S.E., n = 30–61. F, restoration of extracellular Ca2+ (1.5 mm) to cells pretreated with thapsigargin (1 μm) in Ca2+-free HBS fails to evoke Ca2+ entry.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2643496&req=5

fig1: Ca2+ entry evoked by agonists of RyR. A, Ca2+ signals evoked by 4CmC (1 mm) in HBS with or without extracellular Ca2+ and after pretreatment with ryanodine (400 μm, 30 min). Traces (here and in subsequent panels) show the responses from ∼50 individual cells taken from at least three independent experiments (means ± S.E., but with many error bars smaller than the symbols). B, responses to caffeine (1 mm) in normal HBS. C, Ca2+ signals evoked by 4CmC (1 mm) in Ca2+-free medium alone or after pretreatment with thapsigargin (TG, 1 μm). D, peak (after ∼30 s) and sustained (after 200 s) changes in [Ca2+]i evoked by 4CmC in the absence (open bars) and presence of extracellular Ca2+, and the latter after pretreatment (2 min) with nifedipine (Nif, 10 μm) or thapsigargin (1 μm). E, Mn2+ entry (0.5 mm) was measured by quenching of Fura 2 fluorescence in control cells (Q1). Mn2+ was then removed and the cells incubated with thapsigargin (1 μm) for 5 min (during the break between the traces) before again measuring Mn2+ entry (Q2) in the presence of thapsigargin alone (black) or with 4CmC (1 mm, red). Typical traces are shown. Summary data show the slope of the fluorescence change during the second addition of Mn2+ (Q2, with the stimulus) as a % (Q2/Q1) of the slope recorded during the first addition (Q1, without stimulus). Results are means ± S.E., n = 30–61. F, restoration of extracellular Ca2+ (1.5 mm) to cells pretreated with thapsigargin (1 μm) in Ca2+-free HBS fails to evoke Ca2+ entry.
Mentions: Quenching of fura 2 fluorescence by Mn2+ entry was measured in similarly loaded cells bathed in Ca2+-free HBS supplemented with 0.5 mm MnCl2 and with fluorescence recorded after excitation at 360 nm. For all experiments, a basal rate of quench (Q1) was measured before stimulation, and the rate was again measured after stimulation (Q2). The duration of the exposure to MnCl2 (100 s) was chosen to ensure that rates of Fura 2 quenching were linear during both assay periods. The effects of stimuli on rates of Mn2+ entry are reported as a Q2/Q1 ratio, with the ratio uniquely determined for each single cell (see Fig. 1E).

Bottom Line: Subcellular fractionation showed that type 3 IP(3) receptors (IP(3)R3) were expressed predominantly in endoplasmic reticulum, whereas RyR2 were present also in plasma membrane fractions.Using RNAi selectively to reduce expression of RyR1, RyR2, or IP(3)R3, we showed that RyR2 mediates both the Ca(2+) entry and the plasma membrane currents evoked by agonists of RyR.We conclude that small numbers of RyR2 are selectively expressed in the plasma membrane of RINm5F pancreatic beta-cells, where they mediate Ca(2+) entry.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom.

ABSTRACT
Ryanodine receptors (RyR) are Ca(2+) channels that mediate Ca(2+) release from intracellular stores in response to diverse intracellular signals. In RINm5F insulinoma cells, caffeine, and 4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca(2+) entry that was independent of store-operated Ca(2+) entry, and blocked by prior incubation with a concentration of ryanodine that inactivates RyR. Patch-clamp recording identified small numbers of large-conductance (gamma(K) = 169 pS) cation channels that were activated by caffeine, 4CmC or low concentrations of ryanodine. Similar channels were detected in rat pancreatic beta-cells. In RINm5F cells, the channels were blocked by cytosolic, but not extracellular, ruthenium red. Subcellular fractionation showed that type 3 IP(3) receptors (IP(3)R3) were expressed predominantly in endoplasmic reticulum, whereas RyR2 were present also in plasma membrane fractions. Using RNAi selectively to reduce expression of RyR1, RyR2, or IP(3)R3, we showed that RyR2 mediates both the Ca(2+) entry and the plasma membrane currents evoked by agonists of RyR. We conclude that small numbers of RyR2 are selectively expressed in the plasma membrane of RINm5F pancreatic beta-cells, where they mediate Ca(2+) entry.

Show MeSH
Related in: MedlinePlus