Ca2+ Conduction by an Amino Acid-Gated Ion Channel Related to Glutamate Receptors

Ever since homologs of mammalian ionotropic Glu receptors (iGluRs) were discovered in plants (Lam et al., 1998; Lacombe et al., 2001), their molecular-level functions have been an intriguing question. Particularly in need of a direct experimental test is the hypothesis that plant Glu receptor-like (GLR) genes encode ligand-gated channels like the Na+/K+/Ca2+-permeable iGluRs, which mediate electrochemical signaling in animal central nervous systems (Traynelis et al., 2010). The hypothesis was suggested to account for the prevalence of nonselective cation currents across plant cell membranes (Demidchik et al., 2002, 2004) and the ionic events including a Ca2+ rise triggered by amino acids in plant cells (Dennison and Spalding, 2000). The Ca2+ permeability aspect of that hypothesis is especially important, because the influx mechanisms that generate cytoplasmic Ca2+ signals are not well defined at the molecular level (McAinsh and Pittman, 2009; Dodd et al., 2010; Spalding and Harper, 2011). Direct assays of GLR transport activities are needed to test the hypothesis, especially because no GLR is similar to an iGluR in the pore region (Davenport, 2002), which typically determines the ion selectivity of Glu receptors (Traynelis et al., 2010). Animal iGluRs can be expressed and studied with voltage-clamp techniques after expression in Xenopus oocytes (Hollmann et al., 1989), but when this was done with an Arabidopsis (Arabidopsis thaliana) GLR, no amino acid-gated activity was observed (Roy et al., 2008). Transplanting the pore region of a GLR into an iGluR followed by expression of the chimeric protein in Xenopus oocytes was successful. In some cases, a plant GLR pore could form a Na+, K+, and Ca2+ conductance when placed in the context of an iGluR protein, indicating that GLRs may function like iGluRs (Tapken and Hollmann, 2008). The case was strengthened by finding that the large, transient membrane depolarization and rise in cytoplasmic Ca2+ triggered by Glu in wild-type plants (Dennison and Spalding, 2000) depended on GLR genes. Specifically, glr3.3 mutants were found to be defective in their response to each of the six different amino acids (Ala, Asn, Cys, Glu, Gly, and Ser) effective in the root apices of wild-type seedlings (Qi et al., 2006). These results genetically linked ionic events, including Ca2+ transport, to GLRs but fell short of demonstrating GLR3.3 to be a Ca2+-permeable channel. This is also a fair assessment of results obtained with glr1.2 mutant pollen tubes (Michard et al., 2011). A previous electrophysiological study of glr3.4 hypocotyls showed that their cells responded poorly to Asn or Ser and the response to Gly was marginally affected (Stephens et al., 2008). Responses to Ala, Cys, and Glu were normal. These results were interpreted to mean that GLR3.4 was sensitive to the Asn, Gly, and Ser subset of amino acid agonists (Stephens et al., 2008). Direct experimental tests of this hypothesis and the Ca2+ permeability of a GLR3.4 channel are presented here. Human embryonic kidney (HEK) cells are a standard functional expression system for mammalian iGluR channels (Chazot et al., 1999), displaying little or no endogenous amino acid-gated channel activity (Zhang and Huganir, 1999). Much important information about the molecular functions of iGluRs has been learned by studying them in HEK cells (Keinänen et al., 1990; Monyer et al., 1992; Dravid et al., 2008). Therefore, we transfected HEK cells with a plasmid carrying a GLR3.4-GFP fusion cDNA. The resulting fusion protein was shown by fluorescence microscopy (Fig. 1A) to adopt a pattern very similar to previously published examples of proteins that reached the plasma membrane of HEK cells (Christopher et al., 2007; Tang et al., 2007; Mikosch et al., 2009). The expression pattern of free GFP is shown for comparison (Fig. 1B). Arabidopsis plants stably transformed with the same cDNA controlled by the 35S promoter to increase signal intensity showed a cellular signal consistent with plasma membrane localization (Fig. 1C). Plasma membrane localization was expected, because glr mutants are defective in ligand-gated ion transport at the plasma membrane (Qi et al., 2006; Stephens et al., 2008). Plasmolysis of leaf cells in the transgenic plants produced Hechtian strands of plasma membrane connecting the protoplast to sites of adherence at the cell wall. GFP signal was apparent in the Hechtian strands and in the plasma membrane surrounding the withdrawn protoplast (Fig. 1D). No evidence of chloroplast localization of GLR3.4 reported in a study that also reported plasma membrane localization (Teardo et al., 2011) was detected, though intense chlorophyll fluorescence could have masked a weak GFP signal. Plasma membrane localization of GLR3.4-GFP expressed in animal or plant cells. A, HEK cells transfected with a plasmid containing GFP fused to the carboxy terminus of the GLR3.4 cDNA (pcDEST47-GLR3.4) showed signal throughout the cytoplasm and at the cell periphery, consistent with synthesis and maturation in the endomembrane system and delivery to the plasma membrane. B, Transfection of HEK cells with free GFP showed signal throughout the cell, including the nucleus. C, Arabidopsis plants stably transformed with GLRL3.4-EGFP driven by the 35S promoter showed signal at the periphery of leaf epidermal cells. D, Plasmolyzed Pro-35S:GLR3.4-EGFP leaf cells show GFP signal in the Hechtian strands of plasma membrane (horizontal arrows) that stretch between attachment sites in the cell wall (stained red with propidium iodide) and the plasma membrane surrounding the osmotically withdrawn protoplast (vertical arrows). Images are representative of at least six independent trials. Scale bar = 20 μm. The HEK cell result (Fig. 1A) indicated that a patch-clamp assay of ion transport across the plasma membrane was worth pursuing. The plant cell results (Fig. 1, C and D) indicated that any HEK cell results could be interpreted as relevant to the plant cell plasma membrane, where electrophysiological studies indicate the proteins function (Qi et al., 2006; Stephens et al., 2008). To test for ion channel function of GLR3.4, HEK cells were transfected with a bicistronic plasmid carrying GLR3.4 cDNA not fused to GFP and a separate open reading frame encoding a fluorescent marker protein. Fluorescent cells were subjected to patch clamp analyses. Whole-cell ionic currents across the plasma membrane were measured continuously with the membrane potential clamped at −70 mV. Switching to an Asn-containing bath solution induced a large inward current that sustained without significant desensitization over many seconds, returning to the pretreatment level upon washout (Fig. 2A). Cells transfected with a plasmid carrying only GFP did not respond to this treatment (Fig. 2A). The current trace in Figure 2B shows that Gly was approximately 50% as effective compared with Asn. Because the system displayed little desensitization, each test response could be normalized to that triggered by a strong Asn treatment administered to the same cell, thus accounting for any potential differences in GLR3.4 expression levels between cells. Figure 2C shows the magnitude of the response to each amino acid previously found to be an effective agonist in the wild-type plant (Qi et al., 2006; Stephens et al., 2008) plus the ineffective Phe relative to a preceding 1,000 µm Asn response. The GLR3.4 agonist specificity indicated by impaired electrophysiological responses of glr3.4 mutants (Stephens et al., 2008) was confirmed by the behavior of GLR3.4 expressed in HEK cells. Asn and Ser were strong agonists, Gly less so, and the others not significantly different from Phe. Glu occupies the top position in the desensitization hierarchy in planta, probably by activating GLR3.3, but was a relatively poor agonist of GLR3.4 receptor channels, also as predicted by the earlier mutant studies (Stephens et al., 2008). Figure 2D shows that 10 µm of agonist produced a small response, which increased strongly over the concentration range previously found to be effective in plants (Dubos et al., 2003; Meyerhoff et al., 2005; Qi et al., 2006; Stephens et al., 2008). Across this concentration range, Asn and Ser were similarly strong agonists. Agonist profile of GLR3.4 expressed in HEK cells shown by whole-cell patch clamping. A, Asn-induced inward current across the plasma membrane of a HEK cell expressing GLR3.4 is represented as a downward deflection. No such response was observed in untransfected control cells. Voltage was clamped at −70 mV. B, Sequential exposures to different amino acids in an A-B-A pattern showed a lack of GLR3.4 desensitization and permitted quantification of relative agonist effectiveness. Voltage was clamped at −70 mV. C, Magnitude of amino acid-induced current relative to a preceding response evoked by 1 mm Asn. Each determination of relative effectiveness was performed on a separate cell and each result plotted is the mean ± sem of three or more separate experiments. D, Concentration dependence of current response induced by Asn, Ser, or Glu relative to the response subsequently invoked by 1,000 µm Asn in the same cell. The plotted data are means ± sem, n = 3 different cells at each concentration. The main ions in the solutions employed were Na+ (140 mm inside/140 mm outside), Cl− (140 mm inside/140 mm outside), and Ca2+, which was either 2 mm or 20 mm outside and buffered at sub-micromolar levels inside by EGTA. To identify which of these ions was transported by GLR3.4, current-voltage (I-V) analyses were performed with the whole-cell patch clamp technique following the method used to establish the Ca2+ permeability of N-methyl-d-aspartate-type and kainate-type iGluR channels (Mayer and Westbrook, 1987; Egebjerg and Heinemann, 1993). Figure 3A shows currents during three selected voltage steps from a holding potential of −70 mV before, during, and after the Asn treatment. Subtracting either the pretreatment or post-treatment current from the Asn-treated current at each clamp voltage produced a difference curve that represented the I-V characteristics of the Asn-gated conductance (Fig. 3B). Whether pre- or post-treatment data were used as the reference state, the results were substantively the same, because there was very little desensitization of the system. With 2 mm CaCl2 in the bath, inward currents larger than 100 pA were observed over much of the voltage range, even at 0 mV, where Na+ and Cl− would be in equilibrium based on their equal inside and outside concentrations (Fig. 3B). The difference I-V curve appeared to reverse (I = 0 pA) at 100 mV, approaching the equilibrium potential for Ca2+ (127 mV if [Ca2+] inside is 0.1 µM), indicating a high selectivity for Ca2+ over Na+ or Cl−, both of which have equilibrium potentials of 0 mV. Increasing extracellular CaCl2 to 20 mm in the continuous presence of Asn doubled the inward current over most of the voltage range, as expected only if Ca2+ were the predominantly permeable ion. A positive shift in reversal potential expected after switching to higher external CaCl2 concentration was not observed. Instead, the curve reversed at 75 mV. Still far more positive than the Na+ or Cl− equilibrium potentials and therefore indicative of a high Ca2+ permeability, the negative shift in reversal potential indicated that the GLR3.4 channel was less selective for Ca2+ over Na+ in 20 mm Ca2+ than in 2 mm Ca2+. Following the analytical methods of either Lewis (1979) or Lee and Tsien (1984), selectivity for Ca2+ over Na+ was calculated to drop from 5 × 104 to 7 × 102 when external Ca2+ was raised from 2 mm to 20 mM. Cells transfected with a vector not containing GLR3.4 responded very little to Asn or to increasing CaCl2 concentration; the difference I-V curves reversed near 0 mV (Fig. 3B). Each individual trial summarized in Figure 3B is presented in Supplemental File S1. Ca2+ permeability of GLR3.4 channels expressed in HEK cells shown by whole-cell patch clamping and live-cell Ca2+ imaging. A, Whole-cell current traces recorded from a HEK cell expressing GLR3.4 subjected to a voltage-step protocol before (black), during 2 mm Asn treatment (orange), and after agonist washout (blue). The voltage across the membrane was clamped at values ranging from −140 to +85mV in 15-mV increments for 100 ms each, with a 0.8-s recovery period at −70 mV between steps, but only three steps were selected for display. The magnitude of the current reversibly gated by agonist (difference current) is indicated by arrows. B, Difference current-voltage relationship of GLR3.4-expressing or control cells bathed in a NaCl-based buffer containing 2 or 20 mm CaCl2. Control cells were transfected with the YC3.6 Ca2+ indicator only. The plotted data are means ± sem, n = 5 for control, n = 6 for GLR3.4 transfected cells. C, Cytoplasmic Ca2+ rise triggered by Asn in HEK cells expressing GLR3.4 and the optical Ca2+ sensor YC3.6 or only YC3.6. The 535:480 nm fluorescence ratio, a direct measure of cytoplasmic Ca2+ concentration, rose transiently upon application of 500 µm Asn only in cells expressing GLR3.4. The values are means ± sem, n ≥ 3. Above the plots are confocal microscope images falsely colored to represent the magnitude of the FRET signal at each pixel at selected time points in a representative series. D, Cytoplasmic Ca2+ rise triggered by Gly in HEK cells expressing GLR3.4 and the optical Ca2+ sensor YC3.6. Three independent trials are shown. The above results indicate that GLR3.4 encodes a Ca2+-permeable, Asn/Ser,Gly-gated ion channel. This conclusion was independently tested by monitoring cytoplasmic Ca2+ concentration. The fluorescent marker protein separately encoded by the transfection vector was the YC3.6 Ca2+ indicator protein, permitting cytoplasmic Ca2+ concentration to be monitored by measuring the ratio of YC3.6 fluorescence intensity at two wavelengths with a confocal microscope. Figure 3C shows that Asn triggered a large, transient rise in cytoplasmic Ca2+ concentration in HEK cells expressing GLR3.4 but not in cells expressing only the YC3.6 Ca2+ sensor. Figure 3D shows similar transient responses to Gly in three separate cells expressing GLR3.4. Cytoplasmic Ca2+ concentration rose and returned to baseline within approximately 1 min after amino acid treatment, as observed in the plant (Dennison and Spalding, 2000; Dubos et al., 2003; Meyerhoff et al., 2005; Qi et al., 2006). The patch clamp results show that GLR3.4 channel activity was sustained, not transient. Presumably, active Ca2+ transport systems reset the resting levels in the HEK cell as in the plant (Spalding and Harper, 2011). Unlike the results in Figure 2, which could be internally normalized, Figure 3 results are subject to variability in GLR3.4 expression levels between individual cells, as shown in Figure 3D. The results in Figures 2 and 3, along with previous in planta results (Qi et al., 2006; Stephens et al., 2008), indicate that GLR3.4 is activated by amino acids to admit Ca2+ into the cytoplasm to create a transient rise in Ca2+ concentration. In the years since GLR genes were discovered in plants (Lam et al., 1998; Lacombe et al., 2001), research has focused on their phylogeny and evolution (Chiu et al., 1999, 2002; Turano et al., 2001), expression patterns and transcriptional responses (Chiu et al., 2002; Meyerhoff et al., 2005; Roy et al., 2008), roles in carbon to nitrogen balance (Kang and Turano, 2003), abscisic acid sensing (Kang et al., 2004), control of root growth (Li et al., 2006; Miller et al., 2010), and contributions to ionic relations including Ca2+ signaling (Kim et al., 2001; Dubos et al., 2003; Meyerhoff et al., 2005; Kang et al., 2006; Qi et al., 2006; Cho et al., 2009). Slowest to accrue has been information about the intrinsic transport properties of a GLR protein (Dietrich et al., 2010). Heterologous expression followed by voltage-clamp analysis is typically the most reliable source of this information. As a result of that approach in the current study, the evidence indicating that GLRs encode Ca2+ channels is now independently 2-fold: mutating a GLR impairs amino acid-gated Ca2+ influx and membrane depolarization in the plant (Qi et al., 2006; Stephens et al., 2008) and adding a GLR to a HEK cell creates an amino acid-gated Ca2+ conductance capable of generating a cytoplasmic Ca2+ signal (Figs. 2 and 3). Substantiating a Ca2+-permeable channel in plants with these two independent forms of evidence is significant because Ca2+ signals regulate numerous physiological and developmental processes, yet components of the generative mechanisms are insufficiently understood at the molecular level (McAinsh and Pittman, 2009; Dodd et al., 2010; Spalding and Harper, 2011). Now there seems to be little room for doubt that GLR3.4 and probably other family members function as amino acid-gated, Ca2+-permeable channels that are capable of generating a cytoplasmic Ca2+ signal. This may be the extent of the similarity between plant GLRs and animal iGluRs. GLR3.4 is activated by at least three structurally diverse amino acids (Asn, Ser, and Gly) and thus displays an agonist profile broader than any iGluR. This may be because of the different evolutionary origins of the amino of GLRs and iGluRs et al., 2001), which in the case of GLRs may amino acid sites and the poor selectivity between of iGluRs is not by GLR3.4, which is shown to be selective for Ca2+ over Na+ in standard HEK cell The pore of GLRs probably account for this difference and may to about the of Ca2+ selectivity in Because selectivity of GLR3.4 can be by external Ca2+ concentration (Fig. on near the to the or by (Mayer and Westbrook, research selectivity in ionic similar to in the plant A in Ca2+ selectivity over Na+ in Glu receptors has been observed before et al., The was suggested to Ca2+ influx less on in extracellular Ca2+ concentration and 1993). No physiological of this to plants can be that GLR3.4 probably more than to of the 20 mm external Ca2+ used to Ca2+ permeability in Figure The 20 different GLR encoded in the Arabidopsis could form containing a GLR3.4 is predicted to be gated by Asn, Gly, or Ser as a result of the (Fig. and the earlier finding that in planta responses to these same three amino acids are impaired by glr3.4 (Stephens et al., 2008). can be tested now that HEK cells have been as a system for studying GLR The to and test could be selected on the of of expression in the plant (Chiu et al., 2002; Roy et al., et al., 2010). or transport displayed by channels may about their physiological functions in the in which the GLR3.4 agonists Asn, Ser, and Gly could be significantly in the and therefore effective is means of generating about physiological Asn and Ser and other amino acids are by plant GLR3.4 and other members of the GLR family are expressed in (Chiu et al., 2002; Roy et al., et al., 2010). amino acids that the are to be by et al., and the of and creates amino acid patterns in the that as signals by Ca2+-permeable GLRs to root or The GLR3.4 agonists Asn and Ser and other amino acids are also in the at concentrations that on carbon and nitrogen Because the is in with the GLRs could to cells Ca2+ of glr mutant that of agonist concentrations or patterns may higher level physiological functions of amino acid-gated, Ca2+-permeable The Arabidopsis for GLR3.4 is for were obtained from the of Arabidopsis (Arabidopsis thaliana) were and on containing and and mm to with and with GLR3.4 cDNA was from by reverse the for reverse and with the following with and sites within The resulting was into vector following the protocol was with and and into the mammalian expression vector in which the GFP was with generating the resulting GLR3.4 used for HEK cell To generate the fusion the cDNA of GLR3.4 was as above the following at The in was at the of the The resulting was into the vector followed by a into mammalian expression vector for transfection of HEK or the 102 plant expression vector et al., in which the fluorescent protein was with for fluorescence to the The method was used to the mutant with the cells were in with 100 of and 100 of cells were to 1 before transfection and placed in a with and were performed 6 Transfection Cells were after HEK cells were in buffered solution to min to to any amino HEK cells expressing the GLR3.4 were by the YC3.6 marker with the fluorescence microscope on which the patch clamp was based microscope with a Cells a strong fluorescence signal were selected for whole-cell patch clamp currents were recorded after the whole-cell an patch clamp and a controlled by The currents were at 5 and at 10 The solution 1 10 and and to with with or without the indicated amino The solution 10 5 1 and 10 to with To Ca2+ the solution was to 5 2 or 20 10 to with cells transiently expressing the on were in 1 of the solution agonist was as a to the 1 to the indicated concentration. The YC3.6 signal of nm was at 1 the of a confocal microscope. The FRET at the in a region of within the cell were to the amino acid-induced Ca2+ A confocal microscope was used to in leaf epidermal cells and in cells. with propidium for 5 min the plant cell The were with the and channel was used to the of and propidium leaf epidermal were in for 5 to 20 min before the microscopy was employed were a or a cells on and transiently transfected with the vector were As a control, the vector was The following are in the of this Supplemental File S1. I-V data used to generate Figure ionotropic Glu receptor embryonic kidney current-voltage