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Ca2+ Entry Through Plasma Membrane IP3 Receptors

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Science  14 Jul 2006:
Vol. 313, Issue 5784, pp. 229-233
DOI: 10.1126/science.1125203

Abstract

Inositol 1,4,5-trisphosphate receptors (IP3Rs) release calcium ions, Ca2+, from intracellular stores, but their roles in mediating Ca2+ entry are unclear. IP3 stimulated opening of very few (1.9 ± 0.2 per cell) Ca2+-permeable channels in whole-cell patch-clamp recording of DT40 chicken or mouse B cells. Activation of the B cell receptor (BCR) in perforated-patch recordings evoked the same response. IP3 failed to stimulate intracellular or plasma membrane (PM) channels in cells lacking IP3R. Expression of IP3R restored both responses. Mutations within the pore affected the conductances of IP3-activated PM and intracellular channels similarly. An impermeant pore mutant abolished BCR-evoked Ca2+ signals, and PM IP3Rs were undetectable. After introduction of an α-bungarotoxin binding site near the pore, PM IP3Rs were modulated by extracellular α-bungarotoxin. IP3Rs are unusual among endoplasmic reticulum proteins in being also functionally expressed at the PM, where very few IP3Rs contribute substantially to the Ca2+ entry evoked by the BCR.

Most IP3R in most cells are in the endoplasmic reticulum (ER) (13), but IP3-evoked Ca2+ release also occurs from other intracellular organelles (2, 3). Receptors that evoke Ca2+ release from intracellular stores usually also stimulate Ca2+ entry across the PM (1). This is often through store-operated Ca2+ entry (SOC), where depletion of intracellular stores activates a Ca2+-permeable channel in the PM (46). The SOC channel is not itself an IP3R, although IP3R within the ER may interact with it (4). Non-SOC pathways, often regulated by signals derived from diacylglycerol, also contribute to Ca2+ entry (7), but these channels are not formed from IP3R proteins. Cell-surface labeling, immunolocalization, subcellular fractionation, and whole-cell patch-clamp recording (8) have suggested the presence of IP3R in the PM (9). The patch-clamp results are disputed because the most thoroughly characterized current activated by IP3, Ca2+ release–activated current (CRAC) (10, 11), is also activated by store depletion, has properties distinct from IP3R, and is probably activated when IP3R within ER mediate loss of Ca2+ from intracellular stores. The only clear evidence for functional IP3R in the PM comes from cilia of olfactory neurons, but these IP3R differ from those in ER (12, 13). The only IP3-gated channels detected in the PM are thus not obviously related to IP3R in the ER.

SOC evoked by emptying intracellular Ca2+ stores with thapsigargin occurred in cells lacking IP3R (Fig. 1A) (9, 14). The whole-cell current evoked by store depletion in DT40 cells (ICRAC) is likewise independent of IP3R (15). SOC was completely blocked by low concentrations of Gd3+ [half-maximal inhibitory concentration (IC50), 69 ± 9 nM (Fig. 1C)] (5). Antibody to immunoglobulin M (anti-IgM) (5 μg/ml), which stimulates phospholipase Cγ2 through the B cell receptor (BCR), predictably failed to increase the intracellular Ca2+ concentration ([Ca2+]i) in cells lacking IP3R, but it caused release of Ca2+ from intracellular stores and Ca2+ entry in normal DT40 cells (Fig. 1B) (16). The latter was only partially inhibited (55 ± 4%) by a concentration of Gd3+ (300 nM) that abolished SOC (Fig. 1D). Activation of the BCR, but not SOC, stimulates Ba2+ entry and requires IP3R, leading to an earlier suggestion that IP3R might directly mediate Ca2+ entry (9). Our results establish that the Ca2+ entry evoked by the BCR requires IP3R and cannot be mediated by SOC alone (17).

Fig. 1.

Ca2+ entry in DT40 cells with and without IP3R. Ca2+ signals evoked by (A) thapsigargin (0.5 μM, from arrowhead) or (B) anti-IgM (5μg/ml, from arrowhead) in the presence (black) or absence (gray) of extracellular Ca2+ in (a) DT40 wild-type cells and (b) DT40-KO cells. Traces are shown with fluorescence collected at 1.5-s intervals and with SEM shown at each point. (C) Concentration-dependent inhibition of thapsigargin-evoked Ca2+ entry by Gd3+ in DT40 wild-type cells, with inhibition calculated as the difference between the average increase in [Ca2+]i recorded 350 to 450 s after restoration of extracellular Ca2+ in the presence and absence of Gd3+. (D) Ca2+ signals in DT40 wild-type cells evoked by (a) thapsigargin or (b) anti-IgM (each from arrowhead) in the presence (gray) or absence (black) of 300 nM Gd3+; palest lines show responses in absence of extracellular Ca2+. (E) Time course of the Gd3+-insensitive Ca2+ entry evoked by anti-IgM in DT40-R1 cells (fig. S4Ab) compared with that for Po (as percentage of maximum) recorded using perforated patches in the presence of anti-IgM (Fig. 2F). Results are means ± SEM; n ≥ 3.

In whole-cell patch-clamp recordings from wild-type DT40 cells using K+ as a charge carrier, IP3 (10 μM) (16) stimulated opening of cation-selective channels with a slope conductance (γK) of 213 ± 7pS(n = 27) (Fig. 2, A to C). At the peak of their activity, mean channel open (to) and closed (tc) times were 11.0 ± 0.8 ms and 11.1 ± 1.5 ms, respectively. The channel was inactive in the absence of IP3 (Fig. 2A). The effect of IP3 was inhibited by intracellular heparin (100 μg/ml), a competitive antagonist of IP3, and by high extracellular concentrations of 4-aminopyridine (1 mM) or tetraethylammonium (≥10 mM) (fig. S1). Both are blockers of K+ channels but also inhibit IP3R (18). Inhibition of Cl channels had no effect on IP3-activated currents (fig. S1), nor did extracellular Gd3+ (≤1 μM) (fig. S2).

Fig. 2.

Plasma membrane channels activated by IP3. (A) Whole-cell recordings (5 to 6 min after break-in, with K+ as charge carrier) from DT40 wild-type cells held at 0 mV and stepped to –100 mV for current recordings (a) over 15 s, (b) with added IP3 (10 μM), (c) with IP3 and heparin (100 μg/ml), or (d) with adenophostin A (0.5 μM) in pipette solution. (B) Po is shown during stimulation with IP3 (10 μM) during 15-s periods sampled at 1-min intervals after establishing whole-cell recording and with the intracellular free [Ca2+] buffered at 1 nM (solid), 200 nM (open), or 1 μM (gray) bars. Records that included only a single channel are shown. (C) i-V relationship for the IP3-stimulated (filled circles) and adenophostin A–stimulated (open circles) currents. (D) Po measured as in (B), but with 0.5 μM adenophostin A in the pipette. (E) Thapsigargin-evoked (0.5 μM) Ca2+ signals in intact DT40 cells in the absence (gray trace) or presence (black) of extracellular Ca2+. Whole-cell recordings (at –100 mV) are shown at the indicated times (a, b, and c) from cells treated with only thapsigargin (upper traces) or with thapsigargin and IP3 (lower traces). Results are means ± SEM, n ≥ 5. (F) Perforated-patch recordings (at –100 mV) from DT40-R1 cells with Ba2+ as a major charge carrier (a) without and (b) with anti-IgM (5 μg/ml in bath solution), and i-V relationship for the anti-IgM–activated current. (G) i-V relationship for IP3-activated whole-cell current in DT40-R1 cells (at –120 mV) with Ca2+ as the only charge carrier (50 mM CaCl2 in BS, 200 nM free [Ca2+] in pipette solution; details in table S2). Inset shows data extrapolated to the reversal potential. Traces recorded in the (a) absence or (b) presence of IP3 (10 μM in pipette solution).

The maximal open probability (Po) of the PM channels was 0.54 ± 0.03 (n = 7) (Fig. 2B), similar to that observed for IP3R1 expressed in the nuclear envelope from COS (19) or DT40 cells (Fig. 3B and table S1). In >30 recordings from DT40 cells lacking IP3R (DT40-KO) (9, 14), we never detected IP3-evoked currents (Fig. 3A). As expected, IP3 stimulated Ca2+ release from the intracellular stores of DT40 wild-type cells but not from DT40-KO cells (table S1). In all DT40 wild-type cells on which gigaseals were established (n = 135), IP3 stimulated opening of PM channels, but in recordings that lasted up to 20 min, we never detected more than five simultaneous openings and typically detected only one to three (mean: 1.9 ± 0.2). With so few channels, it is unsurprising that we never (n = 19) detected IP3-gated currents in isolated inside-out membrane patches. Nor could we detect biotinylation of PM IP3R in DT40-KO cells stably expressing rat IP3R1 (DT40-R1), in which the total number of IP3R was more than 20 times as much as that in DT40 wild-type cells (table S1 and fig. S6). In perforated-patch recordings of DT40-R1 cells with Ba2+ as a major charge carrier, anti-IgM caused sustained activation of 2.3 ± 0.3 channels per cell (Po = 0.24 ± 0.02, n = 3) (Fig. 3E), and their conductance (γ = 43 ± 6 pS) (Fig. 3F) was the same as that from whole-cell recording with IP3 as the stimulus (table S2). In three similar recordings without anti-IgM, there was no response. Gd3+-insensitive Ca2+ entry (detected with the Ca2+-sensitive indicator, fluo 4) and opening of PM cation channels (in perforated-patch recordings) proceeded with similar time courses after addition of anti-IgM (Fig. 1E). IP3 and the BCR thus stimulate opening of the same PM channels.

Fig. 3.

Ca2+ entry through plasma membrane IP3R1. (A) Whole-cell recordings (at –100 mV) and (B) isolated inside-out nuclear membrane recordings (at –60 mV) from (a and b) DT40-KO, (c) DT40-R1, (d) DT40-R1VI, and (e) DT40-R1GA cells, without (a) or with (b to e) IP3 (10 μM) in the pipette. There was no channel activity in any of the mutant cell lines in the absence of IP3. (C and D) i-V relationship for the IP3-stimulated currents for (C) whole-cell and (D) nuclear membrane recordings from DT40-R1 (filled circles), DT40-R1VI (open circles), and DT40-R1GA (squares) cells. (E) Whole-cell recordings from mouse B cells and DT40-R3 cells with K+ as charge carrier, (a) without or (b) with IP3 (10 μM in pipette solution). (F) i-V relationship for the IP3-activated currents in B cells (filled circles) and DT40-R3 cells (open circles). (G) All-points current amplitude distribution for whole-cell and nuclear recordings at –40 mV from DT40-R3 cells stimulated with 10 μM IP3. (H) Expression of IP3R1 in different cell lines (10 μg protein per lane), determined using Ab1, which recognizes mammalian IP3R1 but not endogenous IP3R of DT40 cells.

An unexpected feature was the delay of several minutes between intracellular dialysis with IP3 and maximal channel activity (Fig. 2B). This is too long to result from diffusion of IP3, but it could reflect slow intervening steps between IP3 binding to an IP3R and activation of PM channels or slow loss of enzymes that degrade IP3. Results with adenophostin A, a nonmetabolized agonist of IP3R (20), support the second possibility. With adenophostin A (0.5 μM) in the pipette, channels with the same properties as those activated by IP3 were activated without detectable latency and remained active for at least 10 min (γ = 210 ± 9 pS, Po = 0.49 ± 0.14, to = 10.6 ± 1.9 ms, tc = 12.7 ± 2.1 ms, n = 8) (Fig. 2, A, C, and D). A much higher concentration of IP3 (100 μM in pipette solution) or another nonmetabolized agonist of IP3R, dimeric IP3 (21), also activated the channels with reduced (IP3, ≤2 min) or undetectable (dimeric IP3) latency. These results establish that IP3 need not be metabolized for it to activate PM cation channels.

Cytosolic Ca2+ biphasically regulates IP3-evoked Ca2+ release from intracellular stores (22). The PM channels activated by IP3 were inhibited when [Ca2+]i was reduced to 1 nM or increased to 1 μM (Fig. 2B). In both cases, channel activity peaked after 5 to 6 min, but Po was reduced from 0.54 ± 0.03 to 0.09 ± 0.04 and 0.02 ± 0.01 (n = 5), respectively. Adenosine triphosphate (ATP) potentiates the effects of IP3 on IP3R (23). Our IP3-activated currents were recorded with 500 μM ATP (but no Mg2+) in the pipette, but in the absence of ATP, Po was reduced by 90% to 0.057 ± 0.04 (n = 5). The requirement for ATP, but not Mg2+, is noteworthy because only MgATP supports the activities of Ca2+ pumps and protein kinases, whereas Mg2+ is not required for ATP to modulate IP3R (23).

DT40-KO cells never responded to IP3, but DT40-R1 cells were responsive. IP3 stimulated release of Ca2+ from the intracellular stores of permeabilized DT40-R1 cells (table S1) and stimulated opening of single channels in the nuclear envelope (γK = 117 ± 5 pS, Po = 0.38 ± 0.06, to = 12.3 ± 1.6 ms, tc = 11.9 ± 2.9 ms) and cation channels in whole-cell recordings with properties (γK = 214 ± 17 pS, Po = 0.52 ± 0.06) (Fig. 3, A to D) indistinguishable from those of DT40 wild-type cells. The similarities included detection of only 1 to 3 PM channels per cell (mean, 1.7 ± 0.2) and a latency of 5 to 6 min before Po peaked. With Ca2+ as the only charge carrier, γCa was 9.1 ± 1.3 pS (Fig. 2G), and again there were only 3.2 ± 0.2 channels per cell (table S2).

In mouse B cells, in which IP3R3 is the major subtype (fig. S3), IP3 invariably activated PM channels (2.0 ± 0.3 channels per cell) with two major K+ conductances (γK = 147 ± 11 and 75 ± 6 pS, n = 8) (Fig. 3, E and F). After expression of rat IP3R3 in DT40-KO cells, IP3 activated channels in the PM (2.6 ± 0.5 channels/cell) and nuclear membrane. IP3R3 displayed subconductances; a major γK in the PM (132 ± 5 pS) and nuclear envelope (130 ± 6 pS) (Fig. 3, F and G) was indistinguishable from that in B cells (147 ± 11 pS). We conclude that small numbers of IP3-activated channels are expressed also in the PM of B cells. Our results demonstrate that IP3 interacts with an intracellular IP3R to stimulate opening of a PM cation channel, but is that channel itself an IP3R?

SOC in DT40 cells is mediated by ICRAC, which is inwardly rectifying, is highly Ca2+-selective (PCa/PNa ∼1000), and has a very low unitary Ca2+ conductance (γCa < 20 fS) (10). The current activated by IP3 is totally different: It is nonrectifying, γCa is much greater (Fig. 2G), and it is not as selective as ICRAC (table S2). PM IP3R are permeable to Ba2+ and Ca2+ (Fig. 2, F and G) and, consistent with previous analyses of IP3R1 in the nuclear envelope (PCa/PK = 4) (19) and lipid bilayers (PBa/PK = 6) (24), they are poorly selective for bivalent over monovalent cations (table S2). Because Ca2+, but not Ba2+, inhibits IP3R (25), it is notable that Po is decreased when Ca2+ is a major charge carrier and reduced further when it is the sole charge carrier but the number of channels detected in the PM is similar whether Ca2+, Ba2+, or K+ is the charge carrier (table S2). Feedback inhibition by cytosolic Ca2+ cannot therefore limit the number of active channels detected in our recordings.

Over a period when thapsigargin (0.5 μM) stimulated SOC in recordings of [Ca2+]i, it had no effect on whole-cell currents or those evoked by IP3 (Fig. 2E). These results and the Gd3+-insensitivity of the channels (fig. S2) establish that the channels activated by IP3 do not result from IP3 causing more complete emptying of intracellular stores and consequent activation of a SOC pathway. To eliminate any possibility that the effects of IP3 might be a consequence of activating IP3R within intracellular stores, we introduced point mutations into the IP3R1 pore. Mutation of V2548 to I (IP3R1VI) increases γK (19), whereas mutation of D2550 to A (IP3R1DA) creates an impermeant channel (26), and mutation of G2547 to A (IP3R1GA) is expected to decrease γ (27). IP3 failed to stimulate Ca2+ release from permeabilized DT40-KO cells or those expressing IP3R1DA (table S1). In inside-out patches from nuclear membrane, IP3 stimulated the opening of channels and γK was increased by 27 ± 4% in IP3R1VI and reduced by 60 ± 15% in IP3R1GA (Fig. 3, B and D). In whole-cell recordings, γK of the PM channels activated by IP3 was increased by 22 ± 8% for IP3R1VI and decreased by 58 ± 9% for IP3R1GA (Fig. 3, A and C, and table S1). In four DT40 lines stably expressing different amounts of IP3R1DA (Fig. 3H and table S1), stimulation of the BCR never evoked Ca2+ signals, IP3 never evoked Ca2+ release from intracellular stores, and in whole-cell recordings we detected no IP3-activated Ca2+ currents (fig. S5). These results are inconsistent with a suggestion that Ca2+ entry evoked by the BCR requires the IP3R but not its functional pore (26).

We introduced an α-bungarotoxin–binding site (16, 28) into the loop linking the final pair of transmembrane domains of IP3R1 close to the pore (Fig. 4A) and expressed it in DT40-KO cells. In whole-cell recordings from these cells, IP3 stimulated the opening of 2.5 ± 0.2 channels per cell, and both their conductance and Po were lower than for normal IP3R (Fig. 4, B and C). The channels were not activated by α-bungarotoxin alone, but in the presence of intracellular IP3, extracellular (but not intracellular) α-bungarotoxin increased both Po and γK (Fig. 4, B and C) without changing the total number of channels detected (3.0 ± 0.3 channels/cell). These results establish that IP3 directly activates an IP3R in the PM and that Ca2+ entry occurs via its pore. The IP3R is unusual in that the same protein is expressed in the ER and the PM and functions in both as an IP3-gated channel. Our results challenge the notion that ER-resident proteins cannot progress to the PM (29).

Fig. 4.

Regulation of plasma membrane IP3R through an extracellular α-bungarotoxin binding site. (A) Structure of IP3R1 containing an α-bungarotoxin (αBgtx) binding site; sf denotes the selectivity filter, and TMD5 and 6 the surrounding transmembrane regions. (B) Whole-cell recordings from DT40-R1αBgtx cells with K+ as charge carrier in the presence of (a and b) αBgtx in bath solution (100 nM) and/or (b and c) IP3 (10 μM) in pipette solution. (C) i-V relationship for the IP3-stimulated current recorded in the presence (filled circles) or absence (open circles) of αBgtx. Summary results for Po and the γK are shown in the histograms (n = 4 and 8 for cells with and without αBgtx, respectively).

Because we invariably detected very few (∼2 per cell) PM IP3R, despite considerable differences in overall levels of IP3R expression (Fig. 3H and table S1), trafficking of IP3R to the PM is probably precisely regulated. Indeed DT40-R1 cells expressed >20 times as much IP3R as did DT40 wild-type cells, but both cell lines express only ∼2 IP3R at the PM (table S1). Furthermore, although the intracellular stores of cells with more IP3RVI were more sensitive to IP3, the number of IP3-gated channels in the PM never exceeded that in DT40 wild-type cells (table S1). In DT40 wild-type cells, the density of IP3R at the PM is only ∼3% of that in the nuclear envelope, and <0.5% of all IP3R are expressed at the PM (fig. S6). Nevertheless, two IP3R with the properties revealed by our whole-cell recordings would allow more than sufficient Ca2+ entry to cause the Gd3+-insensitive Ca2+ signal detected after activation of the BCR (fig. S6).

The same IP3R1 has almost twice the γK when expressed in the PM relative to the nuclear envelope (table S1), and although a major γK for IP3R3 was similar in both membranes, the distribution between subconductance states differed (Fig. 3G). Different membranes may selectively stabilize different subconductances of the IP3R, such that the same IP3R behaves differently in the two membranes.

We conclude that most IP3R are expressed in intracellular stores but that a tiny fraction is reliably directed to the PM, where they contribute substantially to the Ca2+ entry evoked by the BCR. Different Ca2+-regulated processes are likely to respond very differently to Ca2+ dribbling into the cell from huge numbers (possibly >10,000) of ICRAC channels (10) or gushing into the cell through only two IP3R.

Supporting Online Material

www.sciencemag.org/cgi/content/full/313/5784/229/DC1

Materials and Methods

Figs. S1 to S7

Tables S1 to S3

References

References and Notes

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