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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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Caffeine and 4CmC activate the same large-conductance channels in the plasma membrane of RINm5F cells and pancreatic β-cells. A and B, cell-attached (A) and excised patch (B) recordings from RINm5F cells with cesium methanesulfonate in both BS and PS at a holding potential of -100 mV. Caffeine (1 mm) or 4CmC (1 mm) were included in BS as indicated. In the bottom trace of each panel, cells were preincubated with ryanodine (400 μm, 30 min) before stimulating with caffeine. Traces are representative of ≥ 5(n = 2 for ryanodine) independent experiments (see “Results and Discussion”). Arrows denote closed state. For each recording, the boxed area is shown on an expanded timescale on the right. C, current-voltage relationship for channels activated by caffeine and 4CmC. Results are means ± S.E. n = 6–9 (most error bars are smaller than the symbols). D, typical excised patch recording from a rat pancreatic β-cell with cesium methanesulfonate in both BS and PS, at a holding potential of -40 mV, and with 4CmC (1 mm) included in BS as indicated. The boxed area is shown on an expanded timescale on the right. E, corresponding current-voltage relationship for β-cells (means ± S.E., n = 5).
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fig3: Caffeine and 4CmC activate the same large-conductance channels in the plasma membrane of RINm5F cells and pancreatic β-cells. A and B, cell-attached (A) and excised patch (B) recordings from RINm5F cells with cesium methanesulfonate in both BS and PS at a holding potential of -100 mV. Caffeine (1 mm) or 4CmC (1 mm) were included in BS as indicated. In the bottom trace of each panel, cells were preincubated with ryanodine (400 μm, 30 min) before stimulating with caffeine. Traces are representative of ≥ 5(n = 2 for ryanodine) independent experiments (see “Results and Discussion”). Arrows denote closed state. For each recording, the boxed area is shown on an expanded timescale on the right. C, current-voltage relationship for channels activated by caffeine and 4CmC. Results are means ± S.E. n = 6–9 (most error bars are smaller than the symbols). D, typical excised patch recording from a rat pancreatic β-cell with cesium methanesulfonate in both BS and PS, at a holding potential of -40 mV, and with 4CmC (1 mm) included in BS as indicated. The boxed area is shown on an expanded timescale on the right. E, corresponding current-voltage relationship for β-cells (means ± S.E., n = 5).

Mentions: Plasma Membrane Channels Activated by Agonists of RyR—In cell-attached patch-clamp recordings from RINm5F cells with cesium methansulphonate in both the bathing solution (BS) and pipette solution (PS), concentrations of 4CmC (1 mm) or caffeine (1 mm) typical of those used to activate RyR (41) stimulated channel activity (Fig. 3A). Pre-incubation (30 min) with a high concentration (400 μm) of ryanodine, sufficient to inhibit all RyR subtypes (50), abolished responses to both stimuli (Fig. 3A). Similar results were obtained with excised patches (Fig. 3B). In both sets of recordings, GΩ seals were obtained in about 50% of attempts. The frequency with which channels were detected varied between 20 and 80% in different cell preparations. It is difficult to estimate reliably the average number of channels in a patch: too many large-conductance channels may prevent formation of a GΩ seal, and patches with undetected channels will include those that failed to respond for a variety of experimental reasons. However, most active patches included one or two active channels, and very few had 3–4 channels: the average number of channels per excised patch was 1.8 ± 0.2. An analysis that assumes a random distribution of channels between patches likewise suggests the presence of ∼2 channels/patch (supplemental Table S4). Assuming that our patch-clamp recordings are from ∼20% of the total PM, these results suggest the presence of fewer than ∼10 of these channels in the PM of each RINm5F cell. It proved impossible to achieve reliable whole cell recordings.


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

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

Caffeine and 4CmC activate the same large-conductance channels in the plasma membrane of RINm5F cells and pancreatic β-cells. A and B, cell-attached (A) and excised patch (B) recordings from RINm5F cells with cesium methanesulfonate in both BS and PS at a holding potential of -100 mV. Caffeine (1 mm) or 4CmC (1 mm) were included in BS as indicated. In the bottom trace of each panel, cells were preincubated with ryanodine (400 μm, 30 min) before stimulating with caffeine. Traces are representative of ≥ 5(n = 2 for ryanodine) independent experiments (see “Results and Discussion”). Arrows denote closed state. For each recording, the boxed area is shown on an expanded timescale on the right. C, current-voltage relationship for channels activated by caffeine and 4CmC. Results are means ± S.E. n = 6–9 (most error bars are smaller than the symbols). D, typical excised patch recording from a rat pancreatic β-cell with cesium methanesulfonate in both BS and PS, at a holding potential of -40 mV, and with 4CmC (1 mm) included in BS as indicated. The boxed area is shown on an expanded timescale on the right. E, corresponding current-voltage relationship for β-cells (means ± S.E., n = 5).
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Related In: Results  -  Collection

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fig3: Caffeine and 4CmC activate the same large-conductance channels in the plasma membrane of RINm5F cells and pancreatic β-cells. A and B, cell-attached (A) and excised patch (B) recordings from RINm5F cells with cesium methanesulfonate in both BS and PS at a holding potential of -100 mV. Caffeine (1 mm) or 4CmC (1 mm) were included in BS as indicated. In the bottom trace of each panel, cells were preincubated with ryanodine (400 μm, 30 min) before stimulating with caffeine. Traces are representative of ≥ 5(n = 2 for ryanodine) independent experiments (see “Results and Discussion”). Arrows denote closed state. For each recording, the boxed area is shown on an expanded timescale on the right. C, current-voltage relationship for channels activated by caffeine and 4CmC. Results are means ± S.E. n = 6–9 (most error bars are smaller than the symbols). D, typical excised patch recording from a rat pancreatic β-cell with cesium methanesulfonate in both BS and PS, at a holding potential of -40 mV, and with 4CmC (1 mm) included in BS as indicated. The boxed area is shown on an expanded timescale on the right. E, corresponding current-voltage relationship for β-cells (means ± S.E., n = 5).
Mentions: Plasma Membrane Channels Activated by Agonists of RyR—In cell-attached patch-clamp recordings from RINm5F cells with cesium methansulphonate in both the bathing solution (BS) and pipette solution (PS), concentrations of 4CmC (1 mm) or caffeine (1 mm) typical of those used to activate RyR (41) stimulated channel activity (Fig. 3A). Pre-incubation (30 min) with a high concentration (400 μm) of ryanodine, sufficient to inhibit all RyR subtypes (50), abolished responses to both stimuli (Fig. 3A). Similar results were obtained with excised patches (Fig. 3B). In both sets of recordings, GΩ seals were obtained in about 50% of attempts. The frequency with which channels were detected varied between 20 and 80% in different cell preparations. It is difficult to estimate reliably the average number of channels in a patch: too many large-conductance channels may prevent formation of a GΩ seal, and patches with undetected channels will include those that failed to respond for a variety of experimental reasons. However, most active patches included one or two active channels, and very few had 3–4 channels: the average number of channels per excised patch was 1.8 ± 0.2. An analysis that assumes a random distribution of channels between patches likewise suggests the presence of ∼2 channels/patch (supplemental Table S4). Assuming that our patch-clamp recordings are from ∼20% of the total PM, these results suggest the presence of fewer than ∼10 of these channels in the PM of each RINm5F cell. It proved impossible to achieve reliable whole cell recordings.

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