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* This work was supported by the Wellcome Trust and the Biotechnology and Biological Sciences Research Council (UK). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The online version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S6. 1 Both authors contributed equally.
Ryanodine receptors (RyR) are Ca2+ channels that mediate Ca2+ 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 Ca2+ entry that was independent of store-operated Ca2+ entry, and blocked by prior incubation with a concentration of ryanodine that inactivates RyR. Patch-clamp recording identified small numbers of large-conductance (γK = 169 pS) cation channels that were activated by caffeine, 4CmC or low concentrations of ryanodine. Similar channels were detected in rat pancreatic β-cells. In RINm5F cells, the channels were blocked by cytosolic, but not extracellular, ruthenium red. Subcellular fractionation showed that type 3 IP3 receptors (IP3R3) 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 IP3R3, we showed that RyR2 mediates both the Ca2+ 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 β-cells, where they mediate Ca2+ entry.
), and the three subtypes of RyR are each directly regulated by Ca2+. However, RyR differ in whether their most important physiological stimulus is depolarization of the plasma membrane (RyR1), Ca2+ (RyR2) or additional intracellular messengers like cyclic ADP-ribose. The latter stimulates both Ca2+ release and insulin secretion in β-cells (
), and where RyR or IP3R are expressed in other membranes it may allow them to affect membrane potential.
Both Ca2+ entry and release of Ca2+ from intracellular stores contribute to the oscillatory increases in cytosolic Ca2+ concentration ([Ca2+]i) that stimulate exocytosis of insulin-containing vesicles in pancreatic β-cells (
). The importance of this sequence is clear from the widespread use of sulfonylurea drugs, which close KATP channels, in the treatment of type 2 diabetes. Ca2+ uptake by mitochondria beneath the PM further stimulates ATP production, amplifying the initial response to glucose and perhaps thereby contributing to the sustained phase of insulin release (
). RyR and IP3R are thus implicated in mediating responses to each of the major physiological regulators of insulin secretion: glucose and incretins.
Here we report that in addition to expression in intracellular stores, which probably include both the ER and secretory vesicles and/or endosomes, functional RyR2 are also expressed in small numbers in the PM of RINm5F insulinoma cells and rat pancreatic β-cells.
Cell Culture—RINm5F cells were cultured at 37 °C in humidified air containing 5% CO2 in RPMI 1640 medium containing l-glutamine (2 mm), fetal calf serum (10%), Hepes (1 mm), and 2-mercaptoethanol (50 μm). Cells were passaged every 3–4 days when confluent.
Isolation of Rat Pancreatic β-Cells—Pancreatic β-cells were isolated from 3-week-old, male Wistar rats by Dr. Noel Smith (Department of Clinical Biochemistry) (
). Briefly, the pancreas was perfused with Hank's balanced salt solution (HBSS, Sigma) containing collagenase P (1 mg/ml, Roche), excised and incubated at 37 °C for 15 min. The islets were then washed in cold HBSS (600 × g, 5 min) and purified using a Histopaque discontinuous gradient (Histopaque, 1.077 g/ml; Histopaque 1, 1.119 g/ml; Sigma). Dispersed cells were resuspended in RPMI medium and allowed to attach to poly-d-ornithine-coated Petri dishes for patch-clamp recordings from isolated cells.
Subcellular Fractionation—Confluent cells (1.2 × 106 cells) were washed twice with ice-cold phosphate-buffered saline (PBS: 136.9 mm NaCl, 2.7 mm KCl, 1.5 mm KH2PO4, 7.7 mm Na2HPO4, pH 7.4), scraped into 5 ml of the same medium, centrifuged (650 × g, 5 min) and resuspended in 850 μl of 0.3 m sucrose supplemented with Hepes (10 mm, pH 7.4), benzamidine (1 mm), NaN3 (2 mm), protease inhibitors (1 Roche Applied Science complete protease inhibitor mini-tablet/10 ml) and dithiothreitol (5 mm). After homogenization (15 strokes in a Dounce homogenizer) and then passage through a 25-gauge needle (30 times), the homogenate was centrifuged (1000 × g, 10 min, to give pellet (P1) and supernatant (S1) fractions). The supernatant was then re-centrifuged (12,000 × g, 10 min, to give P2 and S2). Sucrose media were prepared in 100 mm KCl, 5 mm MgCl2, 100 mm imidazole, pH 6.8. Layers of 8.5% (1 ml), 40% (1.5 ml) and 60% (1.5 ml) sucrose were successively layered beneath 750 μl of the S2 supernatant fraction. The gradient was centrifuged (100,000 × g, 2 h, Beckman Coulter SW55T rotor), and fractions (0.25 ml, 1–19) were collected from the top of the gradient.
RNAi-mediated Knockdown of RyR1, RyR2, and IP3R3—Cells (5 × 105 cells/well) grown in 6-well plates for 2 days were transfected using Lipofectamine RNAimax with a mixture of two RNAi for either RyR1, RyR2 (10 nm each, Invitrogen), or IP3R3 (10 nm, Qiagen). After 36–48 h, cells were re-seeded (5 × 105 cells/well) and after a further 48 h transfected again with the same RNAi mixture. Cells were used 48–60 h after the second transfection. The RNAi used are listed in supplemental Table S1.
Immunoblotting and Protein Assays—Cells (3 × 35-mm dishes) were washed twice in PBS and lysed (PBS with 1% Triton X-100). Protein concentrations were measured using the Quant-iT protein assay kit (Invitrogen). After SDS-PAGE (3–8%, Invitrogen NuPAGE), proteins were transferred to a polyvinylidene difluoride membrane using an iBLOT apparatus (Invitrogen) and incubated with primary antibody. The antibodies used were: monoclonal IP3R3 (Transduction Laboratories, 1:1000), RyR2 (Calbiochem, 1:1000), rabbit RyR1 (
) (1:1000), plasma membrane Ca2+-ATPase (Alexis, 1:1000) and rabbit polyclonal calnexin (Santa Cruz Biotechnology, 1:500). The blots were then incubated with horseradish peroxidase-conjugated donkey anti-rabbit (AbCam, 1:5000) or goat anti-mouse (Promega, 1:5000) secondary antiserum and visualized with SuperSignal West Pico chemiluminescent substrate (Pierce). Bands were quantified using GeneTools software (Syngene).
Quantitative PCR—Total RNA was isolated from cell lysates and reverse transcribed into cDNA (20 μl) using a fast lane cDNA direct kit (Qiagen). QPCR was performed on a Rotor-Gene 6000 (Corbett Research) in a final volume of 15 μl using Sensimix Plus SYBR master mix (Quantace), 0.5 μm actin primer or Quantitect primers for IP3R1–3 and RyR1–3, and 0.4 μl cDNA. Primers are listed in supplemental Table S2. QPCR conditions were: initial denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. After the last cycle, a melting curve was performed to confirm the identity of the amplified product. Data were analyzed using quantification software supplied with Rotorgene 6000 (
Single Channel Recording—Cells attached to uncoated plastic Petri dishes were rinsed with, and then incubated in, bath solution (BS) containing 140 mm cesium methanesulfonate, 10 mm Hepes, 500 μm BAPTA, 270 μm CaCl2 (free [Ca2+] = 246 nm), pH 7.1. Where indicated, cesium methanesulfonate was iso-osomotically replaced by KCl, NaCl, Tris-HCl or N-methyl d-glucamine-HCl. Pipettes were pulled from borosilicate glass and had resistances of 10–30 MΩ when filled with pipette solution (PS). Most recordings were performed with symmetrical solutions. Currents were recorded with an Axopatch 200B amplifier using pClamp 9.0, digitized at 10 kHz and filtered at 1 kHz. Capacitative currents were cancelled before recording. Single channel currents were recorded in the cell-attached or inside-out configuration of the patch-clamp technique. All recordings were performed at room temperature. Currents were analyzed using pClamp 9.0 software.
Measurements of [Ca2+]i and Mn2+Entry—Cells cultured on 22-mm round glass coverslips were incubated with Fura 2AM (2 μm prepared in anhydrous DMSO) in HBS supplemented with probenecid (2.5 mm) and Pluronic F127 (0.02%) for 45 min at 20 °C, followed by a further 45-min incubation to allow de-esterification of the indicator. HBS had the following composition: 135 mm NaCl, 5.9 mm KCl, 1.2 mm MgCl2, 11.6 mm Hepes, 11.5 mm glucose, and 1.5 mm CaCl2 (replaced by 1 mm EGTA in Ca2+-free HBS), pH 7.3. Fluorescence ratios (excitation at 340 and 380 nm; emission at 510 nm) from single cells were collected at 20 °C at 5-s intervals. After correction for autofluorescence (by addition of 1 μm ionomycin with 10 mm MnCl2), fluorescence ratios were calibrated to [Ca2+]i using a look-up table created from standard Ca2+ solutions (Molecular Probes).
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).
Materials—Fura 2AM and all culture media, except serum (Sigma), were from Invitrogen. All other reagents, including ryanodine, caffeine, 4CmC, and ruthenium red, were from Sigma.
RESULTS AND DISCUSSION
Ca2+Entry Evoked by Agonists of RyR—In single cell measurements of [Ca2+]i in RINmF5 insulinoma cells, most cells responded to caffeine (1 mm) or 4-chloro-m-cresol (4CmC, 1 mm) with a substantial increase in [Ca2+]i (Fig. 1, A and B). A lower concentration of 4CmC (0.5 mm), sufficient to activate RyR1 and RyR2, but not RyR3 (
), evoked a similar sustained Ca2+ signal (Δ[Ca2+]i = 275 ± 18 and 176 ± 11 nm, for 1 mm and 0.5 mm 4CmC, respectively). Treatment with a high, inactivating concentration of ryanodine (400 μm) evoked no Ca2+ signal itself, but abolished the subsequent response to 4CmC (Fig. 1A). Although each stimulus was effective in the absence of extracellular Ca2+, both the peak amplitude and sustained responses were substantially attenuated in Ca2+-free medium (Fig. 1, A–D).
Pretreatment of cells in Ca2+-free medium with thapsigargin (1 μm, 2 min) to empty the intracellular Ca2+ stores that express the SR/ER Ca2+-ATPase (SERCA) massively reduced the 4CmC-evoked increase in [Ca2+]i, but without abolishing it (Fig. 1C). The small residual Ca2+ release is probably mediated by RyR in either secretory vesicles (
). Quenching of fura 2 fluorescence evoked by unidirectional Mn2+ entry across the PM was also stimulated by 4CmC (Fig. 1E). These results establish that in RINm5F cells, agonists of RyR stimulate both release of intracellular Ca2+ stores (from both ER and secretory vesicles or endosomes) and Ca2+ entry. Others have also reported that agonists of RyR evoke Ca2+ release and Ca2+ entry in both β-cells and insulinoma cells (
). Thapsigargin (1 μm) did not cause a substantial increase in [Ca2+]i, but it clearly emptied intracellular stores because it almost abolished the Ca2+ release evoked by 4CmC (Fig. 1C). However, thapsigargin caused no detectable Ca2+ entry. The small responses to thapsigargin were similar in the presence and absence of extracellular Ca2+ (not shown), and restoration of extracellular Ca2+ to thapsigargin-treated cells failed to evoke a Ca2+ signal (Fig. 1F). Under similar conditions, the same stocks of thapsigargin stimulated both Ca2+ release and SOCE in HEK cells (not shown). Furthermore, prior-treatment of RINm5F cells with thapsigargin neither evoked unidirectional Mn2+ entry nor affected the Mn2+ entry evoked by 4CmC (Fig. 1E). We conclude that depletion of intracellular Ca2+ stores fails to evoke significant SOCE in RINm5F cells. The lack of detectable SOCE is consistent with reports from insulinoma (
) where SOCE was either very small or undetectable.
Although agonists of RyR evoke Ca2+ release (Fig. 1, B and C) and SOCE is apparently absent from RINm5F cells (Fig. 1, E and F), both the initial and sustained responses to caffeine (not shown) or 4CmC (Fig. 1D) were substantially reduced in the absence of extracellular Ca2+. Blocking voltage-gated L-type Ca2+ channels with nifedipine (10 μm) had no significant effect on either the peak or sustained responses to 4CmC, and nor had thapsigargin any effect on the sustained Ca2+ signals evoked by 4CmC (Fig. 1D).
Depolarization with 25 mm KCl (by replacement of NaCl) caused an increase in [Ca2+]i (by 291 ± 12 nm, n = 48) that was substantially blocked (∼60%) by 10 μm nifedipine (not shown). Under these depolarizing conditions and with nifedipine (10 μm) present, 4CmC (1 mm) evoked a further increase in [Ca2+]i of 169 ± 21 nm (n = 46). The latter suggests that although 4CmC may lead to some activation of L-type Ca2+ channels (note the small, but statistically insignificant, inhibition of the peak 4CmC-evoked Ca2+ signal by nifedepine in Fig. 1D), these channels can mediate no more than a very small component of the 4CmC-evoked Ca2+ signal.
The results so far establish that agonists of RyR evoke Ca2+ (or Mn2+) entry that is independent of SOCE or voltage-gated Ca2+ channels. Another study also showed that a RyR-selective agonist stimulates Ca2+ entry via a SOCE-independent pathway in β-cells, although it speculated that it resulted from RyR-mediated release of Ca2+ from a non-ER store causing activation of Ca2+-permeable TRP (transient receptor potential) channels in the PM (
). Our subsequent experiments show that the Ca2+ entry is mediated directly by RyR in the PM.
Differential Distributions of RyR and IP3R—The quantitative PCR (QPCR) analysis shown in supplemental Table S3 and the immunoblots shown in Fig. 2A, establish that RyR2 and IP3R3 are the major intracellular Ca2+ channels in RINm5F cells. These results are consistent with previous reports demonstrating that IP3R3 (
IP3R3 and RyR2 were similarly distributed between the supernatant and pellet fractions after crude fractionation (Fig. 2B). Although these simple analyses failed to separate PM and ER membranes, the results are consistent with the substantial presence of IP3R3 and RyR2 within intracellular Ca2+ stores. However, sucrose-gradient centrifugation of a fraction (S2 in Fig. 2B) that included 20% of all IP3R3 and RyR2, clearly separated PM from ER, and IP3R3 from RyR2. IP3R3 appeared in the same fractions as calnexin, an integral ER protein, whereas RyR2 was concentrated in the same fractions as the plasma membrane Ca2+-ATPase (PMCA, Fig. 2, B and C). These results do not prove that RyR2 are expressed in the PM, but they strongly suggest that at least some RyR2 are expressed in a different membrane compartment to IP3R3.
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 (
), 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.
In symmetrical cesium methane sulfonate, the single channel slope conductance (γ) measured in cell-attached patches was the same whether they were stimulated with caffeine (γ = 147.7 ± 4.5 pS, n = 6) or 4CmC (γ = 148.3 ± 4.2 pS, n = 9) (Fig. 3C). We conclude that caffeine and 4CmC stimulate the same ryanodine-sensitive large-conductance cation channels in the PM of RINm5F cells.
Under similar recording conditions (symmetrical cesium methanesulphonate), 4CmC (1 mm) also activated channels in the PM of freshly isolated rat pancreatic β-cells (Fig. 3D). The slope conductance of the major conductance of these channels (γ = 151.5 ± 6.3 pS, n = 5, Fig. 3E) was similar to that in RIN cells.
Properties of Ryanodine-activated Channels in the Plasma Membrane—In excised inside-out patches, a low concentration of ryanodine (4 nm) activated channels that were unaffected by nifedipine (10 μm, not shown) or clotrimazole (Fig. 4A); the latter at a concentration (50 μm) sufficient to inhibit KATP and Ca2+-activated K+ channels (
). The major conductance of the ryanodine-activated channels (γCs = 147.8 ± 3.7 pS, n = 7) was the same as that of the channels activated by caffeine and 4CmC (Fig. 4B). The current-amplitude histograms consistently suggested the presence of minor sub-conductance states with amplitudes of ∼30–50% that of the main conductance (Fig. 4C). Such subconductance states are common features of RyR activated by ryanodine (
). From patches with a single channel, the maximal open probability (Po = 0.2–0.3) was similar for caffeine (1 mm), 4CmC (1 mm) and ryanodine (4 nm) (Fig. 4D), although the mean channel open time (τo) was significantly longer for ryanodine (Fig. 4E). The latter is consistent with the established ability of low concentrations of ryanodine to cause long-lasting openings of RyR (
Ruthenium red is an antagonist of RyR, but it is membrane-impermeant. By first activating channels with 4CmC in the cell-attached configuration and then excising the patch into BS containing ruthenium red (100 μm), we demonstrated that cytosolic ruthenium red rapidly inhibits channel activity. Po in the cell-attached configuration was 0.25 ± 0.02 (n = 20), and remained at 0.24 ± 0.04 after excision into normal BS, but fell within a few seconds to 0.005 ± 0.01 after excision into BS containing ruthenium red (Fig. 5A). These results provide substantial further evidence that the 4CmC-activated cation channels in the PM have the properties expected of RyR.
In excised patches stimulated with 4CmC (1 mm) under symmetrical ionic conditions, the channels were permeable not only to Cs+ (γCs = 148.3 ± 4.2 pS, n = 9), but also to K+ (γK = 169.0 ± 4.8 pS, n = 5) and Na+ (γNa = 130.2 ± 2.1 pS, n = 4) (Fig. 5, B and C). A similar number of channels and with similar Po was detected under each of these conditions (not shown). The channels were impermeable to N-methyl-d-glucamine (NMDG) chloride (Fig. 5B). Our attempts to measure Ba2+ and Ca2+ conductances were frustrated by resealing of membrane around the patch-pipette. We were, therefore, unable to determine the relative permeability of the PM channels for monovalent and bivalent cations. Comparison of γ for the PM channels with analyses of RyR2 reconstituted into lipid bilayers (
) shows that after correction for the different ionic conditions, γ of the PM channels is consistently 3–4-fold lower than for RyR2 in bilayers (supplemental Table S5). Our previous analysis of IP3R expressed in the PM or nuclear envelope also suggested that exactly the same channel can have very different γ when expressed in different membranes (
). A similar explanation may account for the different γ of RyR2 in the PM and bilayers.
We have shown that RyR2 are expressed in RINm5F cells (Fig. 2A) and that their distribution is consistent with a presence in the PM (Fig. 2, B and C). A variety of agonists of RyR (caffeine, 4CmC, ryanodine) activate both Ca2+ entry in intact cells and poorly selective large-conductance cation channels in the PM. The responses are blocked by antagonists of RyR (ryanodine, ruthenium red), but not by antagonists of other cation channels. Our final experiments use RNAi to confirm that RyR2 directly mediate these responses.
Inhibition of RyR2 Expression Inhibits Responses—Using RNAi, we selectively reduced expression of RyR1, RyR2 or IP3R3 by >50%, without reducing expression of the non-targeted proteins (Fig. 6, A and B, and supplemental Table S6).
In excised patch-clamp recordings from mock-transfected cells or cells transfected with RNAi to RyR1, 4CmC-activated cation channels were detected in ∼70% of recordings that achieved a GΩ seal, but the success rate fell to 21 ± 6% for cells treated with RNAi to RyR2 (Fig. 6, C and D). The peak Ca2+ signals evoked by 4CmC, which depend largely on Ca2+ entry (Fig. 1D), were also selectively attenuated by treatment with RNAi to RyR2 (Fig. 6, F and G). Likewise, the effect of 4CmC on Mn2+ entry was almost abolished in cells treated with RNAi to RyR2, whereas RNAi to IP3R3 had no effect (Fig. 6E). We conclude that RyR2 are expressed in the PM and directly mediate Ca2+ entry.
Expression of Functional RyR2 in the Plasma Membrane of RINm5F Cells—We have demonstrated the expression of small numbers (∼10 channels/cell) of functional RyR2 in the PM of RINm5F cells and provided more limited evidence for functional RyR in the PM of rat pancreatic β-cells. Although RyR and IP3R are widely expressed intracellular Ca2+ channels, accumulating evidence suggests that the same channels may also be expressed within the PM of some cells. In B-lymphocytes, for example, very small numbers of functional IP3R (2–3 IP3R/cell) are reliably expressed in the PM, where they mediate about half the Ca2+ entry evoked by activation of the B-cell receptor (
Several studies have detected cation channels with large, although rather variable, conductances that are activated by agonists of RyR in the PM of different cell types. These include osteoblasts, where RyR2 are proposed to mediate extracellular Ca2+-sensing (
). In each case, the density of the PM channels was very low, typically no more than ∼10 channels/cell, and often just 2–3 channels/cell. None of these reports established the molecular identity of the channel and several were reluctant to conclude that it was a RyR (
), but together they provide evidence that small numbers of RyR may be expressed in the PM of several different electrically excitable cells. Our results extend these observations to RINm5F cells and pancreatic β-cells and, more importantly, they unequivocally establish that RyR2 expressed in the PM can directly mediate Ca2+ entry.
These results indicate that in some cells, small numbers of IP3R or RyR can be expressed in the PM and mediate Ca2+ entry. The small number of these channels is significant because both RyR and IP3R have such large conductances (Figs. 3C, 4B, and 5C) that very few channels can mediate substantial Ca2+ entry (
). Selective targeting of RyR (and not IP3R) to the secretory vesicles of β-cells might therefore be one step that contributes to selective expression of small numbers of RyR in the PM.
Abundant evidence implicates RyR in mediating physiological responses of β-cells to glucose and incretins (see Introduction). Our results suggest that their involvement in these responses is likely to involve RyR within both intracellular stores and the PM, and within the latter it is likely that RyR may directly mediate Ca2+ entry and contribute to regulation of membrane potential. It remains unclear whether the non-ER RyR reside predominantly in endosomes or secretory vesicles (
), but it is significant that their presence in either organelle suggests a dynamic relationship between exocytosis of insulin-containing vesicles and trafficking of RyR2 between intracellular organelles and the PM.
We have shown that small numbers of functional RyR2 are expressed in the PM of RINm5F insulinoma cells and rat pancreatic β-cells, where they directly mediate Ca2+ entry and may also regulate membrane potential. We suggest that exocytosis of insulin-containing vesicles and the subsequent retrieval of membrane by endocytosis (