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Departments of Physiology (S.J.P., J.M.-P., M.P.M.-S.) and Pharmacology (E.A.B.), University of Cambridge, Cambridge, United Kingdom
Abstract
Many physiological and pathophysiological situations generate a significant increase in extracellular K+ concentration. This is known to influence a number of membrane conductances and exchangers, whereas direct effects of K+ on the activation of G protein-coupled receptors have not been reported. We now show that Ca2+ release evoked by P2Y1 receptors expressed in 1321-N1 astrocytoma cells is markedly potentiated by small increases in external K+ concentration. This effect was blocked by the phospholipase-C inhibitor U-73122 (1-[6-[[17]-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), but not by its analog U-73343 (1-[6-[[17]-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione), and not by nifedipine, Ni2+, Cd2+, or Gd3+. Thus, K+ enhances D-myo-inositol 1,4,5-trisphosphate-dependent Ca2+ release without a requirement for Ca2+ influx. The cation dependence of this effect displayed the order K+ > Rb+ > N-methyl-D-glucamine+, and Cs+ and choline+ were ineffective. The potentiation by K+ is half-maximal at an increase of 2.6 mM (total K+ of 7.6 mM). K+ caused a reduction in EC50 (2.7-fold for a 29 mM increase) without a change of slope; thus, the greatest effect was observed at near-threshold agonist levels. The response to K+ can be explained in part by depolarization-dependent potentiation of P2Y1 receptors [J Physiol (Lond) 555:61eC70, 2004]. However, electrophysiological recordings of 1321-N1 cells and megakaryocytes demonstrated that K+ also amplifies ADP-evoked Ca2+ responses independently of changes in membrane potential. Elevated K+ also amplified endogenous UTP-dependent Ca2+ responses in human embryonic kidney 293 cells, suggesting that other P2Y receptors are K+-dependent. P2Y receptors display a widespread tissue distribution; therefore, their modulation by small changes in extracellular K+ may represent a novel means of autocrine and paracrine regulation of cellular activity.
Virtually all cells generate a large outward concentration gradient for K+, which is used to regulate the membrane potential and to transport ions or solutes. Although only small amounts of K+ flow across the cell membrane during individual action potentials, it is well established that substantial increases in extracellular K+ concentration ([K+]o) can occur over a sustained period of normal nerve or muscle activation, particularly where diffusion is limited by cellular architecture (Sykova, 1983; Sejersted and Sjo/gaard, 2000). In addition, cellular damage or ischemia will generate substantial, larger increases in [K+]o (Sykova, 1983). Various membrane proteins are known to be stimulated by an increase in external K+, either directly as in Na+,K+-ATPase (Glynn et al., 1970), or as a result of K+-induced membrane depolarization. Indeed, a large increase in external K+ concentration is commonly used as a tool to induce membrane depolarization and to generate Ca2+ influx via voltage-gated Ca2+ channels in studies of excitable tissues. The activation of voltage-gated Ca2+ influx via K+-dependent depolarization is also used physiologically in the adrenal glomerulosa cell as a mechanism of detecting small changes in plasma K+ levels (Spat and Hunyady, 2004). This specialized response to K+ results from a fine tuning of ionic conductances to allow voltage-gated Ca2+ influx, predominantly via T-type Ca2+ channels, to be stimulated by very small changes in membrane potential (Spat and Hunyady, 2004).
Seven transmembrane-spanning G protein-coupled receptors (GPCRs) are the largest family of surface proteins and are involved in the regulation of a wide range of physiological processes. Their activation mechanism is not normally considered to be directly regulated by [K+]o, although recent studies have suggested that a number of GPCRs may be sensitive to changes in the membrane potential (Martinez-Pinna et al., 2005). We now show that increases in extracellular K+, including levels observed under physiological conditions (Sykova, 1983; Sejersted and Sjo/gaard, 2000), markedly potentiate ligand-dependent activation of P2Y receptors. This response occurs in Ca2+-free medium and in the presence of a variety of Ca2+ channel blockers, thus results from modulation of IP3-dependent Ca2+ release without a requirement for Ca2+ influx. We also show that the underlying mechanism is in part independent of changes in membrane potential.
Materials and Methods
Solutions and Reagents. The standard external saline contained 145 mM NaCl, 5 mM KCl, 1 mM MgCl2 10 mM HEPES, 1 mM CaCl2, and 10 mM D-glucose, pH 7.35 with NaOH. For Na+-free saline, NaCl was replaced by an equal concentration of choline chloride. Elevation of K+ or other cations was by equimolar substitution of the CleC salt for NaCl (or choline chloride), except for the experiment shown by the open column in Fig. 3, where K+ was added without substitution. For Ca2+-free saline, CaCl2 was replaced by an equal concentration of MgCl2. In patch-clamp experiments, the pipette saline contained 150 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 10 mM HEPES, 0.05 mM K5fura-2, and 0.05 mM Na2GTP (pH adjusted to 7.2 with KOH). Dulbecco's modified Eagle's medium (DMEM) and G418 (Geneticin) were from Invitrogen (Paisley, UK). K5fura-2, fura-2AM, fluo-3AM, and fluo-4AM were from Molecular Probes (Leiden, The Netherlands). All other reagents were purchased from Sigma Chemical (Poole, Dorset, UK). ADP and 2-methylthio-ADP were treated by incubation with hexokinase and glucose, and ATP and 2-methylthio-ATP were treated with creatine phosphate/creatine phosphokinase, to remove contaminating triphosphate or diphosphate nucleotides, respectively, as described previously (Mahaut-Smith et al., 2000; Tung et al., 2004).
Cell Preparation. 1321-N1 astrocytoma cells, stably transfected (Tung et al., 2004) to express the human P2Y1 receptor (1321-N1-hP2Y1 cells), were grown in DMEM containing 10% fetal bovine serum; 1% penicillin, streptomycin, and amphotericin antibiotic antimycotic solution; and 600 e蘥/ml G418 at 37°C in a humidified atmosphere at 5% CO2. Control experiments confirmed that the ADP-evoked Ca2+ responses in the cell clone used were caused by activation only of P2Y1 receptors. First, the order of efficacy of intracellular Ca2+ concentration ([Ca2+]i) responses was 2-methylthio-ADP > ADP > 2-methylthio-ATP > ATP, that being the known agonist profile of the P2Y1 receptor (Nicholas et al., 1996; Leon et al., 1997). Second, the P2Y1 receptor-specific antagonist MRS-2179 competitively inhibited the ADP-evoked Ca2+ response. Third, untransfected host cells gave no responses to ADP in the concentration range used in this study. HEK 293 cells were grown in DMEM supplemented with high glucose and L-glutamine and containing 10% fetal bovine serum and 1% penicillin, streptomycin, and amphotericin antibiotic antimycotic solution. Megakaryocytes from the femoral and tibial marrow of adult male Wistar rats were prepared for whole cell patch clamp as described in detail previously (Martinez-Pinna et al., 2005).
Intracellular Calcium Measurements in Cell Populations. Population measurements of [Ca2+]i were made using a Flexstation II fluorimeter (Molecular Devices, Wokingham, UK). Cells were grown to a confluent monolayer in 96-well black-walled, clear-bottom Costar microtiter plates (Appleton Woods, Selly Oak, Birmingham, UK). Cells were loaded with fluo-4 by incubation with 2 e fluo-4AM for 45 min at room temperature followed by a single wash. Excitation and emission wavelengths were 488 and 525 nm, respectively, and the emitted light was further filtered with a 515-nm long-pass filter. At the start of each experiment, the cells were bathed in either 200 or 150 e蘬 of saline, for single and double addition experiments, respectively. Agonists, antagonists and high K+ salines were added in 50-e蘬 aliquots. For double addition experiments, the second addition always maintained the agonist/antagonist concentration achieved with the first addition.
Intracellular Calcium Measurements from Single Cells. [Ca2+]i was measured at the single cell level using standard imaging or photometric techniques. 1321-N1-hP2Y1 cells were grown on glass coverslips to 60% confluence. For imaging experiments, cells were loaded with fluo-3 by incubation with 2.5 e fluo-3AM for 45 min at room temperature, followed by a single wash. In photometric experiments, fura-2 was included in the patch pipette, and ratiometric recordings were performed using a Cairn spectrophotometer system (Cairn Research Ltd., Kent, UK), during simultaneous whole-cell patch clamp, as described in detail previously (Martinez-Pinna et al., 2004). Fluorescence imaging was performed on a Zeiss LSM 510 confocal microscope (Carl Zeiss, Welwyn Garden City, UK) with excitation at 488 nm and emission collected at >505 nm. The confocal pinhole was set to measure fluorescence from the entire cell thickness. Images were collected from fields of 15 to 30 cells at a rate of 2 Hz.
Electrophysiology. Conventional whole cell patch-clamp recordings were conducted using an Axopatch 200 series patch-clamp amplifier (Axon Instruments, Foster City, CA). Patch pipettes had filled resistances of 3 to 3.5 M. Megakaryocytes were held under voltage clamp, as described previously (Martinez-Pinna et al., 2005). Membrane potential was recorded from 1321-N1-hP2Y1 cells using the current-clamp (zero current) mode.
Reverse Transcription-Polymerase Chain Reaction. RT-PCR was used to detect mRNA for human P2Y2 and P2Y4 in HEK 293 cells. Total RNA was extracted using the RNeasy mini kit (QIAGEN, Dorking, Surrey, UK), and cDNA was prepared using the Omniscript RT kit (QIAGEN). Forward and reverse oligonucleotide primers were as described previously (Jin et al., 1998). After initial denaturation for 135 s at 95°C, 35 PCR cycles with 5 U/e蘬 Taq polymerase (QIAGEN) were conducted as follows: denaturation at 95°C for 40 s, annealing at 65°C (P2Y4) or 55°C (P2Y2) for 40 s and extension at 72°C for 40 s, followed by 10 min at 72°C. Controls to verify that amplified products were not derived from genomic DNA omitted the reverse transcriptase during the RT step, but they were otherwise identical.
Data Manipulation and Statistics. Experiments shown for single cell recordings are representative of at least five other cells. Fluo-4 and fluo-3 fluorescence signals (f) were expressed as f/f0 ratios to normalize to the fluorescence level at the start of the experiment (f0). Background-corrected fura-2 values of 340/380-nm ratio were converted to [Ca2+]i as described previously (Martinez-Pinna et al., 2005). All experiments were conducted at room temperature (22eC25°C). Data were exported for analysis and fitting of concentration response relationships within OriginLab Origin version 6.0 (OriginLab Corp., Northampton, MA). Data are expressed as the means ± standard error of the mean, with statistical difference assessed using Student's unpaired t test. Statistical significance in the figures is shown at levels of p < 0.05 (), 0.01 (), or 0.005 ().
Results
Potentiation of P2Y1 Receptor-Evoked Ca2+ Release by Extracellular K+. Application of 100 nM ADP to 1321-N1-hP2Y1 cells generated an initial transient (<50 s) increase in [Ca2+]i followed by a constant [Ca2+]i indistinguishable from that of the resting state. Figure 1A shows an example of the response at the single cell level measured by fluorescence imaging. An increase in [K+]o of 30 mM with equimolar reduction in Na+ (final K+ and Na+ concentrations of 35 and 115 mM, respectively) had no effect in the absence of agonist (Fig. 1B), demonstrating the lack of intrinsic K+ dependence and thus also voltage-dependent Ca2+ influx or release under these conditions. However, the same increase in [K+]o induced substantial [Ca2+]i transients in the presence of ADP (Fig. 1B). This response was specifically caused by the increase in K+, not the simultaneous decrease in Na+, since no change in [Ca2+]i was observed if 30 mM Na+ was replaced by choline+ (Fig. 3 and Ability of Other Cations to Modulate the ADP-Evoked Ca2+ Response). Potentiation of ADP-dependent Ca2+ responses by an increase of extracellular K+ was still observed in Ca2+-free medium (Fig. 1C), and thus results from release of internally stored Ca2+ rather than activation of latent Ca2+ channels or reversed Na+/K+/Ca2+ exchange. Potentiation of P2Y1 receptor Ca2+ responses by a 30 mM increase in [K+]o was also observed in salines in which all Na+ was replaced with choline+ (Fig. 1D). This rules out an involvement of the Na+,K+-ATPase, for example via changes in internal Na+, because the K+ dependence of this pump is saturated at a [K+]o of 5 mM under Na+-free conditions (Glynn et al., 1970). It is noteworthy that enhancement of P2Y1 responses was observed after smaller increases in [K+]o, even 1.5 mM (Fig. 1E), which is equivalent to the shift in [K+]o that has been estimated to occur in skeletal muscle T-tubules under physiological conditions (Sejersted and Sjo/gaard, 2000). A 1.5 mM increase in [K+]o evoked a single [Ca2+]i transient, whereas the response to a 30 mM increase was more robust, often causing multiple Ca2+ spikes (compare Fig. 1, B and E). However, because of significant heterogeneity in the magnitude of the Ca2+ response to 100 nM ADP, possibly resulting from variability in receptor density, the concentration dependence to the K+ effect was not further examined at the single cell level. Nevertheless, it was of particular interest that K+ could induce a [Ca2+]i increase in some cells that failed to respond to the agonist alone (Fig. 1F). Overall, therefore, the data in Fig. 1 demonstrate that P2Y1 receptor responses are markedly potentiated by small increases in [K+]o within the concentration range that cells will experience under physiological and pathophysiological conditions (Sykova, 1983; Sejersted and Sjo/gaard, 2000).
Extracellular K+ Decreases the EC50 for ADP at the P2Y1 Receptor. To further characterize the effect of K+ on P2Y1 receptors, we measured average ADP-evoked [Ca2+]i increases in 1321-N1 cells using a Flexstation II 96-well fluorimeter. The concentration-response curve for the ADP-stimulated peak [Ca2+]i increase was shifted to the left by an increase in [K+]o, without a significant change in maximum response or slope (p > 0.05; Fig. 2A). The average EC50 for ADP was shifted 2.7-fold by a 29 mM increase in [K+]o (53 ± 8 nM, n = 6, in 5 mM K+; 20 ± 4 nM, n = 6, in 34 mM K+; p < 0.05). Thus, as observed at the single cell level, the most dramatic enhancement of P2Y1 responses by K+ occurred at threshold concentrations of ADP (for example, 10 nM; Fig. 2B). Increased [K+]o potentiated P2Y1 receptors in a concentration-dependent manner (Fig. 2C), with half-maximal enhancement of the standard response in normal saline after an increase of 2.6 mM K+ (total [K+]o level of 7.6 mM). K+ also caused a concentration-dependent potentiation of P2Y1 receptors when increased from a starting level of zero, in which case a half-maximal effect was observed at 4.2 mM (not shown). For Fig. 2, A to C, ADP was premixed with high K+ saline; however, K+ also enhanced the average P2Y1 response when increased after the initial agonist-evoked [Ca2+]i increase (Fig. 2D, trace 1), as described above at the single cell level (Fig. 1). The lack of effect of saline addition in the presence of the agonist (Fig. 2D, trace 2), or of either saline addition or elevation of K+ in the absence of agonist (Fig. 2D, trace 3), confirms that mechanical release of nucleotides (Lazarowski et al., 2000) did not contribute to the responses measured in this 96-well fluorimeter.
Ability of Other Cations to Modulate the ADP-Evoked Ca2+ Response. An increase in external divalent cation concentration (Mg2+ or Ca2+) in the range 1 to 10 mM caused a concentration-dependent decrease in ADP-evoked Ca2+ responses (not shown) as reported previously for P2Y1 receptors in platelets (Hall et al., 1994). However, other monovalent cations could substitute for K+ in the potentiation of the ADP-evoked Ca2+ response in the 1321-N1-hP2Y1 cell (Fig. 3). The ability to enhance the initial Ca2+ increase evoked by 100 nM ADP displayed the order of potency: K+ > Rb+ > NMDG+, whereas Cs+ and choline+ were ineffective when the concentration of each ion was increased by 30 mM with an equimolar decrease in Na+. The lack of effect of Cs+ and choline+ increases suggest that a decrease in external Na+ has little or no role in the response to K+ or other monovalent cations. This was confirmed by the marked enhancement of ADP-mediated Ca2+ responses when K+ was increased by 30 mM without substitution, whereas an additional 30 mM NaCl had no effect (Fig. 3, open columns).
The Potentiation of P2Y1 Receptors by K+ Does Not Require Activation of Voltage-Dependent Calcium Channels. In excitable cells, the main mechanism whereby an increase in extracellular K+ can stimulate a [Ca2+] response is via membrane depolarization and activation of voltage-gated Ca2+ channels. Indeed, in adrenal glomerulosa cells increases in external K+ of only 1 to 2 mM can generate substantial voltage-dependent Ca2+ influx (Spat and Hunyady, 2004). However, in the 1321-N1-hP2Y1 cells, K+ still potentiated the response to ADP in the presence of blockers of voltage-gated Ca2+ channels, including Ni2+ (200 e), Cd2+ (100 e), and nifedipine (10 e) (Fig. 4). At 100 e, La3+ and Gd3+ abolished the ADP-evoked responses in normal and 34 mM K+ (not shown), suggesting that at high concentrations these common tools used to inhibit Ca2+ influx were directly interfering with activation of the P2Y1 receptor. However, the enhancement of the response to ADP by elevated K+ was maintained in the presence of 1 eGd3+ (Fig. 4B), a concentration of this multivalent cation reported to block store-dependent (capacitative) calcium entry (Broad et al., 1999). Together with the observation that the response is present in Ca2+-free medium (Fig. 1C), these data demonstrate that K+ can enhance ADP-dependent activation of P2Y1 receptors via a mechanism independent of Ca2+ influx. The small reduction in [Ca2+] increase evoked by either ADP or ADP/K in the presence of Ni2+,Cd2+, and Gd3+ compared with the control, can be explained by the inhibitory effect of all these ions on store-dependent Ca2+ influx, and thus reduced levels of Ca2+ within the intracellular stores. The ability of K+ to enhance ADP-dependent Ca2+ release in the presence of 10 e nifedipine (Fig. 4, A and B) also rules out a role for dihydropyridine receptors acting directly on G protein-coupled cascades, and thus IP3 production, as shown to occur in skeletal and smooth muscle (Araya et al., 2003; Valle-Rodriguez et al., 2003). Several pieces of evidence, therefore, demonstrate that the effect of K+ on P2Y1 receptor-evoked Ca2+ responses depends not upon activation of voltage-gated Ca2+ channels or other forms of Ca2+ influx but upon the release of Ca2+ from internal stores.
Essential Role for Phospholipase-C, and Thus IP3 Production, in the Responses to ADP and K+. Pretreatment of 1321-N1-hP2Y1 cells for 10 min with 10 e U-73122, a phospholipase-C inhibitor (Smith et al., 1990), abolished the response to both ADP and ADP in high K+ (Fig. 5). In contrast, an identical treatment with the inactive analog U-73343, had no significant effect on the [Ca2+]i increases evoked by ADP and ADP/high K+ (Fig. 5C). This indicates an essential role for activation of phospholipase-C and thus IP3 production in the response to K+. The 1321-N1-P2Y1 cells lacked functional Ca2+-induced Ca2+ release via ryanodine receptors, because 10 mM caffeine failed to generate a Ca2+ response (data not shown). Thus, IP3-dependent Ca2+ release can fully explain the response to ADP and ADP/K+. This is consistent with previous studies in both heterologous and native systems demonstrating that the P2Y1 receptor couples to Ca2+ mobilization via Gq proteins and phospholipase-C (Nicholas et al., 1996; Offermanns et al., 1997; Martinez-Pinna et al., 2005) and suggests that K+ directly enhances P2Y1 receptor-dependent activation of this IP3-generating pathway.
Role of Membrane Depolarization in the Potentiation of P2Y1 Receptors by Extracellular K+. One major effect of an increase in [K+]o is membrane depolarization, which we have shown to directly enhance Ca2+ release evoked by ADP via P2Y1 receptors in the megakaryocyte (Martinez-Pinna et al., 2005, and references therein). 1321-N1 cells readily form electrical connections with their neighbors; therefore, voltage-clamp experiments proved difficult, and we turned to "current-clamp" whole cell patch-clamp measurements combined with single cell photometry to assess the role of membrane potential in the [Ca2+]i response to K+. 1321-N1-hP2Y1 cells held under patch clamp were generally less responsive to ADP compared with the noninvasive conditions used in Figs. 1 and 2, possibly because of mechanically triggered release of ATP/ADP during gigaOhm seal formation and thus partial receptor desensitization. For example, 100 nM ADP usually evoked only a small or negligible [Ca2+]i increase (Fig. 6, A and B). Nevertheless, an increase in K+ still caused a substantial [Ca2+]i increase if applied in addition to the nucleotide (Fig. 6, A and B). For a [K+]o increase of 30 mM, a substantial membrane depolarization (30 ± 5 mV; n = 5) was observed in parallel with the [Ca2+]i increase. This is within the range of depolarizations previously reported to directly potentiate Ca2+ mobilization via P2Y1 receptors in the electrically inexcitable megakaryocyte (Martinez-Pinna et al., 2004). A 1.5 mM K+ increase was also able to mobilize Ca2+ during exposure of 1321-N1-hP2Y1 cells to 100 nM ADP, whereas the membrane potential displayed only a very small (3mV) depolarization. Spontaneous depolarizations of similar or slightly larger amplitude were observed in many cells during exposure to ADP alone without inducing changes in [Ca2+]i (see, for example, Fig. 6B). This suggests that K+ potentiates P2Y1 receptor signaling in part independently of membrane potential shifts. The megakaryocyte is a cell type in which the ADP-evoked [Ca2+]i response depends upon P2Y1 receptors (Martinez-Pinna et al., 2005) and is amenable to whole cell voltage-clamp recordings without incurring major P2Y receptor desensitization (Martinez-Pinna et al., 2004). Application of 30 nM ADP to a megakaryocyte clamped at eC75 mV generated a small oscillatory [Ca2+]i increase followed by a sustained plateau phase (Fig. 6C). Subsequent elevation of [K+]o from 5 to 35 mM without alteration of the membrane potential generated a large [Ca2+]i transient. This effect was also observed in Ca2+-free saline (n = 5; not shown), confirming that K+ enhances ADP-evoked Ca2+ release. Thus, K+ is able to potentiate P2Y1 receptor-evoked Ca2+ mobilization by both voltage-dependent (Martinez-Pinna et al., 2004) and voltage-independent mechanisms.
Extracellular K+ Potentiates Ca2+ Mobilization Stimulated by Other P2Y Receptors. To investigate whether other P2Y receptor subtypes are modulated by K+, we turned to HEK 293 cells, which display robust endogenous Ca2+ responses to UTP. UTP potently stimulates IP3-dependent Ca2+ mobilization via P2Y2 and P2Y4, but not P2Y1 receptors (Nicholas et al., 1996; Leon et al., 1997). RT-PCR revealed the presence of transcripts for P2Y4 (Fig. 7A), but not P2Y2, in the HEK 293 cells used for the present study. This is consistent with a previous quantitative mRNA study on HEK 293 cells (Moore et al., 2001) and with evidence from immunocytochemical and functional studies (Fischer et al., 2003; Wirkner et al., 2004) that the P2Y4 mRNA there encodes the P2Y4 receptor (Moore et al., 2001). An elevation of external K+ in the absence of agonist activated a [Ca2+]i increase because of the presence of endogenous voltage-gated Ca2+ channels (Berjukow et al., 1996), which was entirely blocked by 200 e NiCl2 (Fig. 7B). However, even in the presence of 200 e Ni2+, K+ was able to potentiate the [Ca2+]i response to UTP when added simultaneously with the agonist (particularly at threshold levels of the agonist; Fig. 7C), or if added subsequent to the initial UTP-evoked Ca2+ transient (Fig. 7D, trace 1). As observed for P2Y1-evoked Ca2+ responses, the effect of K+ on the initial response to UTP was caused by a leftward shift in the concentration-response curve without a change in the slope (Fig. 7E). The EC50 shifted 10-fold from 30 ± 4 e in 5 m MK+ to 2.8 ± 3 e in 34 mM K+ (n = 4; p < 0.01), which was more pronounced than the effect on ADP stimulation of 1321-N1-hP2Y1 cells (see above). Furthermore, K+ produced a significant increase in the maximal response to UTP (Fig. 7E), which was not observed for P2Y1 receptors. Together, these data indicate that P2Y4, like P2Y1 receptors are potentiated by increases in extracellular K+ and that this may be a common feature of G protein-coupled nucleotide receptors.
Discussion
The P2Y1 receptor displays a very widespread distribution in adult and developing tissues (Simon et al., 1997; Moore et al., 2001; Cheung et al., 2003). This identified subtype has well established roles in hemostasis and thrombosis (Kunapuli et al., 2003), and evidence is emerging for specific functions in other tissues such as the regulation of gene expression of some synaptic effectors in skeletal muscle (Tsim et al., 2003) and modulation of some neuronal ion channels (Filippov et al., 2004). We now show that physiologically relevant increases in [K+]o significantly potentiate signaling via P2Y1 receptors via a mechanism independent of calcium influx or voltage-gated calcium channels. The synergy that we demonstrate could amplify cellular responses in a number of situations. For example, tissue injury will extensively release K+ from the cytoplasm of damaged cells and may act to accelerate the initial stages of hemostasis by potentiation of platelet P2Y1 receptor responses (Kunapuli et al., 2003). Furthermore, concurrent extracellular increases in both ATP and K+ can occur. For example, in skeletal muscle, ATP is released either with acetylcholine at the neuromuscular junction or from muscle fibers passively when stressed (Schwiebert and Zsembery, 2003) and significant [K+]o increases are known to occur in this tissue, particularly during exercise. Therefore, the effect we observe can be postulated as a link between levels of activity and gene expression or other signaling events in skeletal muscle. Venous K+ can increase to 6mM in moderate exercise and to almost 10 mM during periods of extreme physical activity (Sejersted and Sjo/gaard, 2000). P2Y1 receptors are located in several cardiovascular tissues, including the heart, endothelium, and vascular smooth muscle; the effect we observe here could therefore play a widespread role in adaptations to exercise. P2Y1 receptors are also located in sensory ganglia (Ruan and Burnstock, 2003), and both noxious stimuli and painful injury are known to evoke a sustained increase in [K+]o of up to 3 mM (Svoboda et al., 1988). Therefore, the K+ dependence of the P2Y1 receptor [or other P2Y subtypes expressed in these ganglia (Moriyama et al., 2003) and exhibiting a dependence upon [K+]o] could have relevance to the mechanisms underlying neuropathic pain.
Potentiation of GPCR responses by K+ is not confined to P2Y1 receptors, because a similar effect was observed for UTP-dependent Ca2+ mobilization involving endogenous P2Y4 receptors in HEK 293 cells (Fig. 7). In fact, K+ caused an even greater leftward shift in the dose-response curve for this UTP response compared with ADP activation of P2Y1 receptors (10-fold compared with 3-fold for P2Y1). K+ also enhanced the maximal response to UTP, which was not significantly observed in 1321-N1-hP2Y1 cells and may reflect a greater overall level of amplification for P2Y4 compared with P2Y1 receptors. UTP-sensitive P2Y4 receptors are expressed on a range of neuronal cell types (Ruan and Burnstock, 2003). In addition, P2Y4 has been shown to be expressed on many epithelial surfaces (Suarez-Huerta et al., 2001; Unwin et al., 2003), where large ionic fluxes occur and thus where an elevation of K+ may exert an important regulatory role.
It is well established that small increases in the extracellular K+ concentration, similar to those that we show potentiate P2Y receptors, can generate large increases in intracellular Ca2+ in the adrenal glomerulosa cell (Spat and Hunyady, 2004). The increase in [Ca2+]i leads to release of aldosterone and thus physiological responses to regulate plasma K+ levels. However, the mechanism underlying the response to K+ in the glomerulosa cell contrasts with the effect on P2Y receptors in that it depends upon activation of Ca2+ influx via T-type Ca2+ channels. Synergy is observed between elevated K+ and angiotensin II, although the underlying mechanism is again caused by effects on voltage-gated Ca2+ influx (Spat and Hunyady, 2004).
As a consequence of the leftward shift in the ADP or UTP concentration-response curve, the amplification was particularly pronounced when K+ was added simultaneously with ADP at near-threshold levels of the agonist (Figs. 1F, 2B, and 7B). It was also interesting to note that K+ generated substantial [Ca2+]i increases if added after the initial agonist-evoked transient, when the [Ca2+]i had returned to near resting levels (Figs. 1 and 2D). Furthermore, at the single cell level, the effect of a K+ increase of only 1.5 mM subsequent to the agonist (Fig. 1, E and F) produced an initial [Ca2+]i spike of similar amplitude to that generated by a much higher K+ level (30 mM increase; Fig. 1B). In part, this may reflect the nonlinear highly cooperative nature of the IP3-dependent Ca2+ release from stores (Meyer et al., 1988); however, it may also reflect an ability of K+ to act more effectively on an agonist-bound receptor state.
We have previously shown that Ca2+ signaling via P2Y1 receptors in the megakaryocyte is markedly potentiated by membrane depolarization (Martinez-Pinna et al., 2005). The response is graded with depolarizing pulse amplitude without evidence for a threshold potential (Martinez-Pinna et al., 2004). Thus, the potentiation of P2Y1 receptors by a 30 mM K+ increase, which depolarized the 1321-N1 cells by 30 mV, could in part involve a direct effect of membrane potential. However, an increase of only 1.5 mM K+, which is effective at enhancing P2Y1 receptors in 1321-N1 cells (Fig. 1E), predictably had negligible effects on the membrane potential. In addition, K+ potentiated the ADP (1 e)-evoked [Ca2+]i increase at a constant membrane potential in rat megakaryocytes (Fig. 6), a native cell type where this response is dependent upon the presence of P2Y1 receptors (Martinez-Pinna et al., 2005). Thus, K+ enhances P2Y1 receptor signals via both membrane depolarization and via a more direct effect.
Regarding the underlying mechanism, the complete block of agonist-induced calcium responses by U-73122 in both control cells and cells exposed to elevated K+ is consistent with an effect of the cation at the receptor level leading to calcium mobilization via an IP3-dependent pathway. We can exclude effects of K+ in the range studied here on the relative amounts of the forms of ADP in solution. ADP is largely complexed there with divalent cations as a result of its high affinity for Mg2+ and Ca2+ compared with K+ (stability constants for Mg2+,Ca2+, and K+ binding to ADP have been reported to be 3, 2.81, and 0.67, respectively; Sillen and Martell, 1971). Furthermore, the stability constants for Na+ and K+ are virtually identical (0.65 and 0.67, respectively; Sillen and Martell, 1971); therefore, the standard experimental protocol used in this study, involving equimolar reduction in Na+ with elevation of K+, will not alter the level of free ADP. Indeed, the only changes will be extremely small increases in KADP2eC and decreases in NaADP2eC. A reasonable explanation for the voltage-independent effect of K+ on P2Y1 receptors would be allosteric binding to one or more sites on the exofacial surface. A precedent for such a monovalent cation binding exists in the well established allosteric modulation by intracellular Na+ of several G protein-coupled receptors via binding to a site containing a critical aspartate residue (Horstman et al., 1990). In the present study, the half-maximal value of the K+ concentration dependence was 4.2 or 7.6 mM, for starting concentrations of 0 and 5 mM, respectively, and gives an estimate of the operative K+ affinity. The affinities for K+ and Na+ on many proteins are generally a hundredfold or higher, suggesting a specific K+ binding site at the P2Y1 receptor, as found in a few other well established examples where K+ is functional. For example, the K+ affinity in Shaker K+ channels has been estimated (Thompson and Begenisich, 2001) at 2.7 mM for its high-affinity state when one K+ is in the pore, weakened (allowing fast ion flow) to 65 mM when two ions are there, because of their mutual repulsion and a conformational change (Zhou and MacKinnon, 2003). This K+-chelating site is built from a serine OH and four backbone carbonyls (Zhou and MacKinnon, 2003). A few enzymic proteins also bind an essential K+ ion, some decarboxylases (Toney et al., 1995) and tryptophanase (Isupov et al., 1998), the latter having affinity of 1.4 mM and using, rather similarly, a Glu carboxylate oxygen and four backbone carbonyls. Hence, a K+ binding site on the P2Y1 receptor would be within the known range of functional K+-protein interactions.
In conclusion, we show for the first time that physiologically relevant increases in extracellular K+ significantly potentiate signaling via P2Y receptors. Depolarization can account for part of the response at high, pathophysiological levels of K+; however, the cation also potentiates P2Y1 receptors independently of a change in membrane potential.
Acknowledgements
We thank Gwen Tolhurst and Richard Carter for assistance with RT-PCR, Jon Holdich for advice and assistance with cell culture, Dr. Virgilio L. Lew for helpful comments on the manuscript, and Dr. Dave Thomas for advice on use of the Flexstation Fluorimeter.
doi:10.1124/mol.104.009902.
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