Phosphatidylinositol 4,5-Bisphosphate (PIP2) Modulation of ATP and pH Sensitivity in Kir Channels

A TALE OF AN ACTIVE AND A SILENT PIP2 SITE IN THE N TERMINUS*

Dirk SchulzeDagger, Tobias KrauterDagger§, Hariolf Fritzenschaft, Malle Soom, and Thomas Baukrowitz

From the Institute of Physiology II, Friedrich Schiller University Jena, Teichgraben 8, 07740 Jena, Germany

Received for publication, August 17, 2002, and in revised form, December 2, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Phosphatidylinositol polyphosphates (PIPs) are potent modulators of Kir channels. Previous studies have implicated basic residues in the C terminus of Kir6.2 channels as interaction sites for the PIPs. Here we examined the role of the N terminus and identified an arginine (Arg-54) as a major determinant for PIP2 modulation of ATP sensitivity in KATP channels. Mutation of Arg-54 to the neutral glutamine (R54Q) and, in particular, to the negatively charged glutamate (R54E) impaired PIP2 modulation of ATP inhibition, while mutation to lysine (R54K) had no effect. These data suggest that electrostatic interactions between PIP2 and Arg-54 are an essential step for the modulation of ATP sensitivity. This N-terminal PIP2 site is highly conserved in Kir channels with the exception of the pH-gated channels Kir1.1, Kir4.1, and Kir5.1 that contain a neutral residue at the corresponding positions. Introduction of an arginine at this position in Kir1.1 channels rendered the N-terminal PIP2 site functional largely increasing the PIP2 affinity. Moreover, Kir1.1 channels lose the ability to respond to physiological changes of the intracellular pH. These results explain the need of a silent N-terminal PIP2 site in pH-gated channels and highlight the N terminus as an important region for PIP2 modulation of Kir channel gating.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Kir channels are a superfamily of eukaryotic channel proteins that are expressed in many tissues and responsible for important physiological processes such as cell excitability, insulin secretion, K+ homeostasis, vascular tone, and regulation of the heart rate. Four subunits assemble to a channel. Each subunit contains two transmembrane segments with cytoplasmic N- and C-terminal domains and a connecting loop forming the pore (1). Some members of the Kir channel family are endowed with gating mechanisms such as ATP gating (KATP channels) (2) and pH gating (Kir1.1 and Kir4.1 channels) (3). These gating mechanisms are central for the diverse functions of Kir channels in physiology and the understanding of the related pathophysiology. Kir1, Kir4, and Kir5 channels, that are predominantly expressed in epithelia, are exquisitely sensitive to changes in intracellular pH in the physiological range (3-5). This pH sensitivity is mediated by the protonation of a lysine in the N terminus (Lys-80 in Kir1.1) that induces closure of the channel's pore by an allosteric mechanism (pH gating) (3, 6). Even small changes in the pH sensitivity can cause severe kidney defects such as the Bartter syndrome (3), highlighting the physiological importance of proper pH gating in Kir1.1 channels. Kir6 channels display a very ubiquitous expression pattern and, in coassembly with the sulfonylurea receptor (SUR),1 represent the ATP-sensitive K+ channels (KATP channels) (7). Intracellular ATP closes KATP channels by binding to the Kir6.2 subunits (ATP gating), whereas the SURs act as regulatory subunits endowing the channel with sensitivity to MgADP and pharmacological compounds. The ATP/ADP dependence of KATP channels couples cell metabolism to membrane excitability, which plays an important role in the physiology of many tissues (e.g. pancreas, heart, brain) (1, 2, 8). Highly negatively charged membrane phospholipids, in particular the phosphatidylinositol polyphosphates (PIPs), such as phosphatidylinositol 4,5-bisphosphate (PIP2), were found to interact with Kir channels, and in general they stabilize the open state of the channel (9-14). In addition, PIPs were shown to interfere with the different gating mechanisms of Kir channels. A recent report indicated modulation of pH sensitivity of Kir1.1 channels by PIP2 since a mutation in the C terminus (R188Q) that reduced PIP2 binding also changed the pH sensitivity (15). Further, PIPs are effective modulators of KATP channels because they reduce the sensitivity to inhibition by intracellular ATP (12, 13). The effect on ATP sensitivity is of particular physiological importance since the amount of ATP inhibition determines the activity of KATP channels in cells. Moreover, regulation of PIPs levels via signal transduction pathways represents an effective means to regulate KATP channels by various receptors (12, 13, 16-18). PIP2 was shown to interact with basic residues in the C terminus of Kir6.2. For Kir6.2/SUR channels the basic residues cluster in two regions of the C terminus (176-222 and 303-314) (11-13, 19). Further, two regions in the cytoplasmic C terminus (20, 21) and one region in the N terminus (21) have been identified where mutations markedly reduce ATP sensitivity. Intriguingly, the two C-terminal regions (near 182-185 and 333-338) are in proximity (at least in primary sequence) to the regions that are implicated in PIP2 binding (176-222 and 303-314); however, the mechanistic basis of this coincidence is not clear. The region near Arg-50 in the N terminus has been implicated in ATP inhibition (21), and we mutated, therefore, basic residues in this region (Lys-47-Lys-67) to screen for PIP2 binding sites. This approach identified Arg-54 as an important determinant for PIP2 binding in KATP channels that appeared to be vital for mediating the antagonizing effect of PIP2 on ATP inhibition. Further, we demonstrated that the N-terminal PIP2 site is silent in pH-gated Kir channels (e.g. Kir1.1) because they lack a positively charged residue at the relevant position. Introduction of a positively charged residue (e.g. arginine) largely increased PIP2 binding and, more importantly, impaired pH gating explaining the need of a silent N-terminal PIP2 site for pH-gated Kir channels.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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REFERENCES

Mutagenesis, cRNA Synthesis, and Oocytes Injection-- Murine Kir6.2, rat SUR2A, and Kir.1.1 (ROMK1) were used in this study. Site-directed mutagenesis was performed as described (22) and verified by sequencing. For oocyte expression, constructs were subcloned into the pBF expression vector (23). Capped cRNAs were synthesized in vitro using SP6 polymerase (Promega, Heidelberg, Germany) and stored in stock solutions at -70 °C. Xenopus oocytes were surgically removed from adult females and manually dissected. About 50 nl of a solution containing cRNA specific for SUR2A and Kir6.2 subunits was injected into Dumont stage VI oocytes. Oocytes were treated with collagenase type II (Sigma, 0.5 mg/ml) and incubated at 19 °C for 1-3 days and defolliculated prior to use.

Electrophysiology-- Giant patch recordings (22) in inside-out configuration under voltage-clamp conditions were made at room temperature (~23 °C) 3-7 days after cRNA injection. Neomycin and ATP were purchased from Sigma. L-a-Phosphatidyl-D-myo-inositol-4,5-phosphate (PIP2 from bovine brain) was purchased from Roche Molecular Biochemicals, stored as stocks (1 mM) at -20 °C, diluted in Kint solution to final concentrations, sonicated for 35 min, and used within 6 h. Pipettes used were made from thick-walled borosilicate glass, had resistances of 0.2-0.4 MOmega (tip diameter of 20-30 µm), and were filled with (in mM, pH adjusted to 7.2 with KOH) 120 KCl, 10 HEPES and 1.8 CaCl2. Currents were recorded with an EPC9 amplifier (HEKA electronics, Lamprecht, Germany) and sampled at 1 kHz with analog filter set to 3 kHz. Solutions were applied to the cytoplasmic side of excised patches via a multibarrel pipette and had the following composition in mM (Kint): 100 KCl, 10 HEPES, 2 K2EGTA (total K+ concentration was 120 mM, pH adjusted to 7.2 with KOH). Computational work was done on Macintosh G4 using commercial software (IGOR, WaveMetrics) and Excel 2001 (Microsoft).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Arg-54 in the N Terminus of Kir6.2 Is a Major Determinant for PIP2 Binding-- The region near Arg-50 (Lys-47-Lys-67) was screened for residues that might contribute electrostatically to the binding of PIP2 or ATP (Fig. 1A). Basic residues in this region (Lys-47, Arg-50, Arg-54, Lys-67) were mutated to the negatively charged amino acid glutamate. This charge-reversing substitution should reduce the binding of PIP2 or ATP via electrostatic repulsion if the residue is located close to the respective binding sites. We found that ATP sensitivity was reduced for R50E (as reported previously, (21)) and, surprisingly, increased for R54E, whereas K47E and K67E channels displayed ATP sensitivities similar to WT channels (see below, Fig. 3D). To assess the impact of the different mutants on PIP2 binding we compared the current amplitude briefly after patch excision with that obtained after application of PIP2. This type of assay has been used previously (19) and is based on the finding that PIP2 increases the channel's open probability (Po). For WT channels PIP2 increased the current amplitude (thus Po) only marginally (Figs. 1C and 4B). This suggests that the affinity for PIPs is so high that the endogenous PIP2 already maximally opens the channels. Similar results were obtained for the mutants K47E, R50E, and K67E (Fig. 1C). In distinction, R54E channels showed only little initial activity, but application of PIP2 increased the current amplitude by a factor of 29 ± 9 suggesting a marked reduction in PIP2 affinity (Figs. 1, B and C and 4C). To investigate whether the charge at position 54 is critical, Arg-54 was mutated to a neutral (R54Q) and to a positively charged amino acid (R54K). R54K channels showed WT behavior, whereas the current produced by R54Q channels was increased by a factor of 4.5 ± 1.5 by PIP2 (Fig. 1C). The impact of mutations on PIP2 modulation in the order R54E > R54Q > R54K = WT suggest electrostatic interactions between the charge at position 54 and PIP2. The mutation R176A in the C terminus of Kir6.2 has been previously shown to reduce PIP2 binding (11, 13) and is shown here for comparison. The current amplitude produced by R176A channels was increased by PIP2 by a factor of 3.2 ± 0.5 (Fig. 1C).


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Fig. 1.   Effect of PIP2 on the amplitude of currents mediated by Kir6.2/SUR2A channels and mutants expressed in Xenopus oocytes and studied in giant inside out patches. The same protocol applies to all following experiments (Fig. 1-Fig. 5). A, schematic model of a Kir6.2 subunit residues of interest are highlighted. B, R54E currents measured at -80 mV (inward currents shown as upward deflection), patch excision, and application of 10 µM PIP2 are indicated. C, bars represent the fold change of current amplitude ± S.E. (n > 3) upon application of 10 µM PIP2 for 45 s.

Neomycin Inhibition as an Assay for PIP2 Affinity in Kir Channels-- Neomycin is a polycation that binds specifically to PIP2 and for this reason has been used to determine the PIP2 content in biological membranes (24). In electrophysiological experiments, neomycin was shown to reverse the effects of PIP2 on KATP channels causing inhibition of channel activity and reduction of ATP sensitivity (11, 25). Thus, the neomycin sensitivity of a Kir channel might be a measure of its PIP2 affinity. Accordingly, channels with a high PIP2 affinity are expected to be less sensitive to neomycin than those with low PIP2 affinity. To test this assumption we measured neomycin inhibition of KATP and Kir1.1 channels since Kir1.1 channel are thought to bind PIP2 more tightly than KATP channels. In good agreement, KATP channels (IC50 = 17.1 ± 2.2 µM) were about 3-fold more sensitive to neomycin than Kir1.1 channels (IC50 = 43 ± 10 µM) (Fig. 2C). Using this assay, we tested for the PIP2 affinities of the different N-terminal (K47E, R50E, R54E, R54Q) mutants and the C-terminal mutant R176A of Kir6.2. Some of the mutated channels produced only small currents (R54E, R54Q, R176A) necessitating the measurement of neomycin inhibition subsequent to application of PIP2 (10 µM for 45 s) (Fig. 2A). The mutations R54E, R54Q, and R176A altered neomycin inhibition considerably: the concentration-response curves were much steeper and most of the inhibition occurred between 1 µm and 10 µM (IC50 of about 3 µM) (Fig. 2, A and B). For K47E channels neomycin sensitivity was somewhat increased (IC50 = 38 ± 10 µM) and for R50E channels somewhat reduced (IC50 = 318 ± 183 µM) compared with WT channels (IC50 = 122 ± 98 µM after PIP2) (Fig. 2C), whereas the shape of the concentration-response curves was not changed. These results are qualitatively in good agreement with the findings on PIP2-amplitude modulation (Fig. 1). R54E, R54Q, and R176A largely affected PIP2-amplitude modulation and neomycin sensitivity, whereas K47E and R50E channels showed basically WT behavior.


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Fig. 2.   Neomycin inhibition of Kir1.1, Kir6.2, and mutant channels in inside out patches. A, neomycin inhibition subsequent to application of 10 µM PIP2 for 45 s; neomycin concentration as indicated. B, from data such as shown in A concentration-response curves (CV curves) were obtained and fitted to a standard Hill equation; error bars represent ± S.E. C, from CV curves as in B the IC50 values were determined and plotted as bar with ± S.E. Stars indicate that the IC50 values were determined subsequent to application of 10 µM PIP2 for 45 s.

Arg-54 Determines PIP2 Modulation of ATP Sensitivity in KATP Channels-- The effect of the N-terminal mutations as well as the C-terminal mutant R176A on the PIP2 modulation of ATP inhibition was characterized by comparing the ATP sensitivity before and subsequent to application of 10 µM PIP2 for 45 s. This procedure shifted the IC50 for ATP inhibition of WT channels by a factor of 71 ± 3 (Fig. 3, B, C, and D). Similar values were obtained for K47E (shift factor: 55 ± 17), R50E (shift factor 98 ± 16), and K67E (shift factor 73 ± 15) (Fig. 3C). For R176A the shift factor was reduced to 28 ± 4 (Fig. 3C) in agreement with previous findings on this mutant (13). R54E and R54Q had by far the largest effects on the PIP2 modulation of ATP inhibition with the corresponding shift factors of 2.5 ± 0.3 and 8.2 ± 1, respectively (Fig. 3, A, B, and C). Thus, the mutations R54E and R54Q impaired the modulation of ATP sensitivity by PIP2 with R54E being more potent than R54Q. The impact of R54Q on the antagonistic effect of PIP2 on ATP inhibition is shown directly in Fig. 4A. Application of 50 µM PIP2 readily removed inhibition of WT channel produced by 1 mM ATP (n = 3, Fig. 4A). For R54Q channels even prolonged application (see time scale) of 50 µM PIP2 only marginally antagonized ATP inhibition (n = 3, Fig. 4A). Very similar results were obtained for R54E, whereas R54K showed WT behavior (data not shown). For R176A channels PIP2 modulation of ATP inhibition was significantly reduced; however, clearly less pronounced compared with R54E/R54Q channels (n = 3, Fig. 4A). Fig. 4, B and C shows the effect of PIP2 and ATP on a single WT and R54E channel. WT channels display high initial Po (>0.8) that was only marginally increased upon application of PIP2. Given subsequent to PIP2, 100 µM ATP produced virtually no inhibition (Fig. 4B). In contrast, R54E channels have very low initial Po (<0.05), and PIP2 largely increased the Po to a level similar to WT channels (>0.8); however, 100 µM ATP potently inhibited channel activity (Fig. 4C) in full agreement with the macroscopic currents (Figs. 1 and 3). In summary, these results show that the mutations R54E/R54Q disrupt the ability of PIP2 to antagonize ATP inhibition.


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Fig. 3.   Effects of Kir6.2 mutations on PIP2 modulation of ATP sensitivity. A, R54E currents with applications of 10 µM PIP2 and various ATP concentrations as indicated. B, from data such as in A CV curves were obtained and fitted to a standard Hill equation (13); error bars indicate ± S.E. C, from CV curves in B IC50 values were determined and plotted as bars with ± S.E. D, table with values of the IC50 values before (contr.) and after application of 10 µM PIP2 for 45 s.


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Fig. 4.   Antagonism of ATP inhibition by PIP2 for WT and mutant channels. A, for WT, R54Q, and R176A currents the effect of PIP2 and ATP is shown with concentrations as indicated. Similar results were obtained in at least two further experiments. B, single channel recordings for WT and R54E channels measured at -80 mV and filtered at 2 kHz. Application of PIP2 and ATP as indicated. From the last 3 s before ATP application the Po was estimated to be > 0.8 < 0.9 for WT and R54E. Po was calculated from amplitude histograms (data not shown). The Po of the R54E channels before PIP2 was below 0.05. Similar results were obtained in two further experiments.

Role of the N-terminal PIP2 Site in Kir1.1 Channels-- The arginine at position 54 is highly conserved among the members of the Kir channel superfamily; however, Kir1.1, Kir4.1, and Kir5.1 possess a neutral residue at the corresponding position (Fig. 5A). These channels have in common that they are gated by intracellular protons (3-6, 26). We chose Kir1.1 to study the role of the position Ile-63 that corresponds to Arg-54 in Kir6.2. An Arginine was introduced at position 63 (I63R), and the impact on PIP2 affinity was assayed by monitoring the run down of channel activity upon exposure to a Mg2+-containing solution (Fig. 5B). Mg2+ is thought to induce a run down via a breakdown of PIP2 though activation of phosphatases and lipases associated with the patch (10). WT channels lost most of the channel activity within 12 min. In I63R channels run down was markedly slower indicative of an increased PIP2 affinity (Fig. 5B). Accordingly, the neomycin inhibition of I63R channels was reduced dramatically from an IC50 of 43 ± 10 µM (WT channels) to 7.3 ± 1.7 mM (Fig. 5C).


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Fig. 5.   Effects of I63R on PIP2 affinity and pH sensitivity of Kir1.1 channels. A, sequence alignment of various Kir channels for the region near Arg-54 in Kir6.2; pH-gated channels are indicated with arrows (Alignment was done with the commercial software DNA-Star). B, time course of channel run down for WT and I63R channels induced by application of 2 mM Mg2+; data points are mean ± S.E. C, neomycin inhibition of WT and I63R channels. From experiments such as in Fig. 2A CV curves were obtained and fitted to a standard Hill equation; error bars indicate ± S.E. The IC50 values for WT and I63R were 43 ± 10 µM and 7.3 ± 1.7 mM, respectively. E, currents from WT and I63R channels (F) at the pH values indicated. D, from recordings such as in E and F CV-curves were obtained and fitted to a standard Hill equation (open symbols); error bars indicate ± S.E. pKa and Hill coefficient were 6.8 ± 0.02 and 4.2 ± 0.3 for WT and 5.77 ± 0.08 and 2.2 ± 0.1 for I63R channels. Filled symbols represent CV curves subsequent to application of 20 µM PIP2 for 30 s; pKa and Hill coefficient were 6.79 ± 001 and 2.7 ± 0.3 for WT and 5.75 ± 0.04 and 1.8 ± 0.1 for I63R channels.

To investigate the role of position 63 for pH gating, WT and I63R channels were exposed to various pH values of the bathing solution. Acidification from 7.5 to 6.0 resulted in complete but reversible inhibition of Kir1.1 channels (Fig. 5E). Using a Hill equation the effective pKa and Hill coefficient for pH inhibition were estimated to be 6.83 ± 0.04 and 4.4 ± 0.2, respectively, in good agreement with previous reports (Fig. 5D) (3, 6). The mutation I63R largely reduced the pH sensitivity shifting the pKa to 5.77 ± 0.08 and the Hill coefficient to 2.2 ± 0.1 (Fig. 5, D and F). If this shift in pH sensitivity was caused by an increased binding of PIP2, then application of PIP2 should cause a similar shift in pH sensitivity for WT channels. However, application of PIP2 had no significant effect on pH inhibition (Fig. 5D) suggesting that PIP2 binding was already saturated before the addition of exogenous PIP2. These results point to an effect of I63R on pH gating in Kir1.1 channels that is distinct to the effect on PIP2 affinity (see "Discussion").

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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We screened the proximal N terminus for residues that contribute electrostatically to the binding of PIP2 or ATP by substituting basic residues by the negatively charged glutamate (K47E, R50E, R54E, K67E). Two of these mutations altered ATP inhibition markedly. R50E reduced the ATP sensitivity about 12-fold, whereas R54E increased the sensitivity about 3-fold. However, neither Arg-50 nor Arg-54 are likely to contribute to ATP binding directly as detailed below.

Arg-54 Is an N-terminal Determinant for PIP2 Binding in Kir6.2/SUR Channels-- The mutation R54E resulted in KATP channels with very low Po upon patch excision that was largely increased by addition of exogenous PIP2 (PIP2-amplitude modulation) consistent with a reduced PIP2 affinity. In contrast, WT channels as well as the mutants K47E, R50E, and K67E showed nearly maximal Po upon patch excision, and exogenous PIP2 had little effect. The PIP2 affinity of the different mutant channels clearly correlated with the charge at position 54 with WT and R54K channels having the highest affinity, R54Q channels an intermediate, and R54E the lowest PIP2 affinity. These results strongly suggest electrostatic interaction between Arg-54 and PIP2. Previous work has identified several residues in the C terminus of Kir6.2 as determinants for PIP2 binding, e.g. R176A was shown to reduce PIP2 binding (11, 13, 19). The effect of R176A on PIP2-amplitude modulation was comparable to R54Q (Fig. 1C) suggesting similar importance for the binding of PIP2 to KATP channels. PIP2 binding of WT and mutant channels was also tested using neomycin inhibition as a relative measure for the PIP2 affinity. R54E, R54Q, and R176A channels showed a markedly increased sensitivity to neomycin compared with WT, K47E, and R50E channels. These results further substantiate the view that Arg-54 directly contributes to PIP2 binding.

Arg-54 Plays a Key Role for the PIP2 Modulation of ATP Sensitivity in KATP Channels-- The ATP sensitivity of KATP channels depends on the concentration of PIPs in the membrane. Increasing concentrations of PIP2 increase the IC50 value for ATP inhibition by several orders of magnitude (12, 13), and thus, the shift in ATP sensitivity for a given increase in membrane PIP2 should be related to the PIP2 affinity of the channel. We found that the mutations K47E, R50E, and K67E had virtually no effect on the PIP2 modulation of ATP inhibition in contrast to R54E and R54Q. R54E/R54Q dramatically reduced the effect of PIP2 on ATP inhibition with R54E being more potent than R54Q. The results are in excellent agreement with those on PIP2-amplitude modulation and show that the charge at position Arg-54 determines the effect of PIP2 on Po and on ATP sensitivity.

On the Mechanism of PIP2 Modulation of ATP Inhibition-- As pointed out in the introduction, regions implicated to be important for ATP inhibition and PIP2 binding appear to coincide (at least in primary sequence) in the C terminus of Kir6.2 (19-21). This tendency seems to be even more striking with the identification of Arg-54 as a determinant of PIP2 binding in the N terminus since Arg-50 is an important determinant of ATP inhibition (21). On the mechanistic basis of this finding two explanations come to mind. First, ATP and PIP2 might bind to overlapping sites, and secondly, PIP2 modulates ATP inhibition allosterically by interaction with basic residues in regions that are critical for the gating mechanism that links ATP binding to channel closure. Both alternatives have been put forward recently (27-29). As an argument against physically overlapping binding sites it has been pointed out that mutations in the C terminus that affected PIP2 binding in the most cases did not change ATP inhibition (19). This holds valid also for the N terminus. R50E markedly reduced ATP sensitivity but had no effect on PIP2 modulation of ATP inhibition. On the contrary, R54E largely reduced the effect of PIP2 on ATP inhibition but had no direct effect on ATP sensitivity. Assuming electrostatic repulsion between R54E and PIP2 it is rather unlikely for ATP binding to occur in close proximity to position 54 without sensing the charge at this position. These results argue against overlapping binding sites for PIP2 and ATP in the N terminus and favor an allosteric mechanism. In other words the interaction of PIP2 with Arg-54 appears to disrupt the mechanism that allows ATP to induce channel inhibition. Accordingly, disabling the N-terminal PIP2 site is expected to reduce the ability of PIP2 to modulate the ATP sensitivity as seen with R54E/R54Q channels. The identification of several PIP2 sites in N and C terminus raises the question whether the sites are functionally equivalent. Are there PIP2 sites that preferentially affect ATP inhibition and others that are more important for control of e.g. open-state stability? Comparing R176A and R54Q suggests such functional heterogeneity. Both mutations increased PIP2-amplidude modulation to a similar extent (Fig. 1C) suggesting that Arg-54 and Arg-176 contribute about equally to the effect of PIP2 on open-state stability. However, Arg-176 contributed obviously less to the antagonizing effect of PIP2 on ATP inhibition compared with Arg-54 (Figs. 3C and 4A). Moreover, none of the other potential PIP2 binding sites in the C terminus identified so far appeared to be very critical for the PIP2 modulation of ATP sensitivity. Mutations at these positions produced significant effects on PIP2-amplitude modulation, but no marked effects on the ability of PIP2 to shift ATP sensitivity have been observed (19). These findings suggest a pivotal role of Arg-54 for mediating the antagonizing effect of PIP2 on ATP inhibition.

Role of the N-terminal PIP2 Site for pH Gating in Kir Channels-- The arginine at position 54 is highly conserved among the Kir channel superfamily pointing to a general role for PIP2 binding in Kir channels. Indeed, a recent paper demonstrated that this arginine also contributes to PIP2 binding in Kir2.1 channels (30). Intriguingly, Kir1.1, Kir4.1, and Kir5.1 present with a neutral residue at the corresponding position. A distinctive property of these Kir channels is a strong effect of intracellular protons on the open probability of the channel. This pH gating is mediated by a lysine residue (Lys-80 in Kir1.1) in the N terminus that serves as a pH sensor and is lacking in other Kir channels (3, 6). Protonation of the pH sensor causes reversible inhibition of channel activity in the physiological range. The unusual acidic pKa (approx 6.8) of the lysine is thought to come about from its proximity to two arginines in the N and C terminus of the same subunit forming a "Arg-Lys-Arg triad" that shifts the pKa of the lysine into the physiological range via electrostatic interactions (3).

It has been proposed that PIP2 binding to Kir1.1 alters the pKa for pH gating (15). Thus, we tested whether the absence of the N-terminal PIP2 site might be a prerequisite for Kir1.1 channels to operate in the physiological pH range (6.8-7.5). We observed, indeed, that introduction of an arginine at the N-terminal PIP2 site largely increased the PIP2 affinity of Kir1.1 channels and shifted the pKa for pH inhibition far out of the physiological range (pKa for I63R approx  5.8). It has been shown that reduction in PIP2 binding can shift the effective pKa for pH gating in Kir1.1 channels to more alkaline values. This was based on the finding that low PIP2 concentrations in the membrane or reduction in PIP2 binding, as seen with a mutant channel (R188Q), increased the pH sensitivity (15). We found that PIP2 binding is already saturated for WT Kir1.1 channels since addition of exogenous PIP2 caused no additional shift in the pKa. This outcome does not conflict with previous findings (15); however, it suggests that an increase in PIP2 affinity (e.g. I63R) should not alter pH gating and, thus, should not account for the effect of I63R on pH gating. PIP2 interaction with the N-terminal site might either reduce H+ binding to the pH sensor thus shifting the pKa of lysine 80 (e.g. though a change of the micro environment of Lys-80) or disturb the transduction mechanism allowing protonation of the pH sensor to power channel closure. Indeed, the reduction of the Hill coefficient for I63R (WT = 4.2 ± 0.3 and I63R = 2.2 ± 0.1, Fig. 5D) might indicate reduced coupling between the pH sensor and the gate that controls channel activity. In conclusion, ATP gating in KATP channels and pH gating in Kir1.1 are controlled by a N-terminal PIP2 site. While for KATP channels this site is necessary to allow potent modulation of ATP sensitivity by PIP2, a silent N-terminal PIP2 site is a prerequisite for intact pH gating in Kir1.1 channels. Even subtle changes in the pH sensitivity of Kir1.1 channels cause severe kidney defects as found in patients with the antenatal Bartter syndrome (3). Thus, a high evolutionary pressure to preserve a neutral residue at the N-terminal PIP2 site is expected and consistent with the absence of a positively charged residue in all pH-gated Kir channels.

    ACKNOWLEDGEMENTS

We appreciate the excellent technical support by Dr. Yuan Ruan and thank Dr. Klaus Benndorf and Dr. Bernd Fakler for critically reading the manuscript.

    FOOTNOTES

* This work was supported by Grant Ba 1793 from the Deutsche Forschungsgemeinschaft (to T. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Both authors contributed equally to this work.

§ Present address: flyion GmbH, Waldhäuserstrasse 34, 72076 Tübingen, Germany.

To whom correspondence should be addressed. Tel.: 0049-3641-938860; Fax: 0049-3641-933202; E-mail: thbau@mti-n.uni-jena.de.

Published, JBC Papers in Press, January 4, 2003, DOI 10.1074/jbc.M208413200

    ABBREVIATIONS

The abbreviations used are: SUR, sulfonylurea; PIP, phosphatidylinositol polyphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; WT, wild type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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