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J. Biol. Chem., Vol. 277, Issue 51, 49366-49373, December 20, 2002

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Localization of the ATP/Phosphatidylinositol 4,5 Diphosphate-binding Site to a 39-Amino Acid Region of the Carboxyl Terminus of the ATP-regulated K⁺ Channel Kir1.1^*

Ke Dong, LieQi Tang, Gordon G. MacGregor, and Steven C. Hebert^§

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026

Received for publication, August 23, 2002, and in revised form, October 8, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Intracellular ATP and membrane-associated phosphatidylinositol phospholipids, like PIP₂ (PI(4,5)P₂), regulate the activity of ATP-sensitive K⁺ (K_ATP) and Kir1.1 channels by direct interaction with the pore-forming subunits of these channels. We previously demonstrated direct binding of TNP-ATP (2',3'-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)-ATP) to the COOH-terminal cytosolic domains of the pore-forming subunits of Kir1.1 and Kir6.x channels. In addition, PIP₂ competed for TNP-ATP binding on the COOH termini of Kir1.1 and Kir6.x channels, providing a mechanism that can account for PIP₂ antagonism of ATP inhibition of these channels. To localize the ATP-binding site within the COOH terminus of Kir1.1, we produced and purified maltose-binding protein (MBP) fusion proteins containing truncated and/or mutated Kir1.1 COOH termini and examined the binding of TNP-ATP and competition by PIP₂. A truncated COOH-terminal fusion protein construct, MBP_1.1CC170, containing the first 39 amino acid residues distal to the second transmembrane domain was sufficient to bind TNP-ATP with high affinity. A construct containing the remaining COOH-terminal segment distal to the first 39 amino acid residues did not bind TNP-ATP. Deletion of 5 or more amino acid residues from the NH₂-terminal side of the COOH terminus abolished nucleotide binding to the entire COOH terminus or to the first 49 amino acid residues of the COOH terminus. PIP₂ competed TNP-ATP binding to MBP_1.1CC170 with an EC₅₀ of 10.9 µM. Mutation of any one of three arginine residues (R188A/E, R203A, and R217A), which are conserved in Kir1.1 and K_ATP channels and are involved in ATP and/or PIP₂ effects on channel activity, dramatically reduced TNP-ATP binding to MBP_1.1C170. In contrast, mutation of a fourth conserved residue (R212A) exhibited slightly enhanced TNP-ATP binding and increased affinity for PIP₂ competition of TNP-ATP (EC₅₀ = 5.7 µM). These studies suggest that the first 39 COOH-terminal amino acid residues form an ATP-PIP₂ binding domain in Kir1.1 and possibly the Kir6.x ATP-sensitive K⁺ channels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ATP-sensitive K⁺ (K_ATP) channels couple cell metabolism to K⁺ channel activity (3-7). K_ATP channels are formed by four pore-forming subunits (Kir6.x) in association with four subunits of sulfonylurea receptors (8), SUR1 or SUR2a/b. Kir1.1 has many properties similar to these K_ATP channels and has been suggested to interact with the cystic fibrosis transmembrane conductance regulator or SUR2b to form a glibenclamide-sensitive and ATP-inhibited channel (9, 10). Nucleotides can both inhibit and activate K_ATP channels (3, 4, 11). Inhibition of K_ATP channel activity is mediated by binding of ATP to the Kir subunits, whereas channel activation by ADP occurs upon interaction with the SUR subunits. Phosphatidylinositol phosphates (e.g. phosphatidylinositol 4,5 diphosphate, PIP₂)¹ compete with ATP for gating of K_ATP channels (12-16). The effect of PIP₂ to antagonize the inhibitory action of ATP provides a mechanism for opening K_ATP channels in intact cells in the presence of cytosolic ATP concentrations that would otherwise be inhibitory (13, 16-18).

We recently localized the ATP-binding region to the COOH terminus in each of the three ATP-regulated channels: Kir1.1, Kir6.1, and Kir6.2 (19). We found that maltose-binding (MBP) fusion proteins containing Kir1.1 or Kir6.x COOH termini are efficiently produced in bacteria, are soluble without detergent, and directly bind TNP-ATP with a stoichiometry of at least one ATP per COOH-terminal monomer. In addition, we showed that phosphatidylinositol phospholipids competed for ATP binding to the COOH termini of Kir6.x and Kir1.1 channels (2), providing a mechanism for PIP₂ antagonism of ATP effects.

Kir1.1 or ROMK, originally cloned from rat kidney outer medulla (20), forms the small conductance ATP-regulated K⁺ channel involved in apical K⁺ recycling in thick ascending limb and in K⁺ secretion in principal cells of the mammalian kidney (11). Mutations in the human ROMK gene (Kcnj1) that cause loss of channel function give rise to Bartter's syndrome, which is characterized by volume depletion resulting from renal salt wasting (21, 22). Mice with deletion of the ROMK (Kir1.1) gene exhibit a phenotype consistent with Bartter's syndrome with polyuria, increased urinary Na⁺ loss, and extracellular fluid volume depletion (23). The ROMK-deficient mice have no small conductance channel activity on apical membranes of thick ascending limb or principal cells, consistent with ROMK encoding the small conductance K⁺ recycling/secretory channel in kidney.

In the present study, we further localized the ATP binding within the COOH terminus of Kir1.1. We assessed TNP-ATP binding and its antagonism by PIP₂ to purified maltose-binding fusion proteins with deletions of the cytosolic COOH terminus of Kir1.1. The ATP-PIP₂ binding domain was localized to the first 39 amino acids of the COOH terminus of Kir1.1. We also defined specific arginine residues within this 39-amino acid domain that are critical for TNP-ATP binding. Because these arginine residues are conserved in the Kir6.x channels, similar domains in these channels are likely to also form ATP-binding pockets.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

DNA Constructs and Mutagenesis-- Complementary DNA encoding the COOH terminus of Kir1.1 (amino acids 183-391) was derived from rat Kir1.1a (ROMK1; Ref. 20). The Kir1.1 COOH terminus was subcloned into a XbaI and PstI-digested pMBPT vector (19, 24). The deletions and single amino acid substitutions in the COOH terminus of Kir1.1 were generated using PCR. All full-length and truncated C-terminal fragments of Kir1.1 included a stop codon and were ligated in-frame with MBP. All DNA constructs were verified by PCR and cycle sequencing (Keck Facility, Yale) and then transformed into Epicurian coli® BL21-CodonPlus^TM-RIL competent cells (Stratagene, La Jolla, CA) for the protein expression.

Production and Purification of Maltose-binding Fusion Proteins-- Recombinant proteins were expressed as previously described by us (19). Briefly, 1 liter of Luria-Bertani medium with 0.1 mg/ml ampicillin and 0.2% glucose was inoculated with 10 ml of an overnight culture of BL21 cells expressing the fusion vector and grown to an A₆₀₀ of ~0.5 at 37 °C. Induction was performed with 0.3 mM isopropyl -D-thiogalactoside at 37 °C for 2 h. The cells were harvested and centrifuged at 4,000 × g for 20 m at 4 °C. The cell pellet was resuspended in 50 ml of Column Buffer (in mM: 20 Tris-HCl, 200 NaCl, 1 EDTA, pH 7.4) and frozen overnight at 20 °C. The sample was thawed in ice water and lysed with a probe sonicator (4 times for 30 s, with 30-s intervals) in an ice-water bath. The sample was then centrifuged at 9,000 × g for 30 min at 4 °C. The supernatant was kept and diluted 1:5 with Column Buffer. The diluted extract was loaded into a 25-ml column containing 15 ml of amylose resin and washed with 12 column volumes of Column Buffer. The fusion protein was eluted with Column Buffer plus 10 mM maltose, and 1.5-ml fractions were collected. Protein was detected by UV absorbance at 280 nm, dialyzed against 50 mM Tris-HCl, pH 7.5, and kept at 80 °C until experiments. All MBP fusion proteins were efficiently expressed, except for MBP_1.1C180, and could be purified at milligram quantities (10-25 mg/liter of bacterial culture) without exposure to detergents or denaturing agents. All MBP fusion proteins ran as single bands at their expected molecular masses on 10% PAGE gels.

TNP-ATP Binding-- Binding of ATP to these recombinant fusion proteins was performed generally as described previously (19). Briefly, 5 µM recombinant protein was dissolved in 50 mM Tris-HCl at pH 7.5, and TNP-ATP binding was detected by the increase in fluorescence (Ex = 403 nm; 546 nm; slit widths 5 nm) upon binding to recombinant protein using a Fluromax-3 spectrofluorometer (Jobin Yvon Inc., Edison, NJ). Temperature was maintained at 22 ± 0.1 °C by a circulating water bath (Neslab, Portsmouth, NH). Incremental additions of TNP-ATP were delivered to plastic cuvettes from stock solutions (0.2-1.0 mM). Protein was denatured by addition of 4 M urea where indicated.

The TNP-ATP fluorescence enhancement factor () with binding to proteins, TNP-ATP to protein subunit stoichiometry (N), and dissociation constant (K_d, µM) were determined using a modified version of the binding equation derived by Faller (25) and as modified by us (19) using GraphPad PRISM^TM 3.0 software. As seen in Equation I, observed fluorescence intensity (F_obs) in arbitrary units (a.u.) is given by

(Eq. 1)

where P is the protein concentration (µM). Q and Q₂ are constants (fluorescence intensity per µM or µM² of free TNP-ATP, respectively) derived independently from the concentration dependence of TNP-ATP fluorescence intensity in buffer alone (F_Buffer) and account for the inner filter effect (25): F_buffer = Q[TNP-ATP] + Q₂[TNP-ATP]². Q_c is the slope of the F_Buffer versus (TNP-ATP) curve in buffer alone as shown in Equation 2.

(Eq. 2)

Protein light scatter intensity (F_{light scatter}) was subtracted from all F_obs values.

was measured at a fixed TNP-ATP concentration of 5 µM by determining the increase in F_obs with increasing protein concentration. The F_obs data were fit well by a single exponential and F was determined as F_obs at infinite protein concentration when all TNP-ATP would be bound. The enhancement factor was then calculated as shown in Equation 3.

(Eq. 3)

As shown in Equation 4, using individual  values we calculated the concentrations of free (F) and bound (B) TNP-ATP as described by Moczydlowski and Fortes (26).

(Eq. 4)

(F) is then the difference between total (TNP-ATP) and (B). Bound versus free TNP-ATP plots were analyzed using a standard binding model that follows mass action as seen in Equation 5,

(Eq. 5)

where B_max is the maximal TNP-ATP binding. Data were also plotted for Scatchard analysis as described (19).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Localization of the TNP-ATP Binding Segment in the Kir1.1 COOH Terminus-- The concentration-dependence relationships of TNP-ATP fluorescence (F_obs) with full-length (MBP_1.1C-WT) and deletion mutants of the Kir1.1 COOH terminus are shown in Fig. 1A. The numbers associated with the construct names in Fig. 1A indicate the length of the deletion from the carboxyl end of the COOH-terminal construct. F_obs increased in a non-linear, concentration-dependent manner with all COOH-terminal deletions from 84 to 180 amino acid residues. TNP-ATP fluorescence increased above that seen for MBP_1.1C-WT with deletion of 84 residues (MBP_1.1CC84). In contrast, further truncations of the distal COOH terminus up to 136 residues (MBP_1.1CC136) reduced maximal fluorescence with 10 µM TNP-ATP below that of MBP_1.1CC84 and to the fluorescence of MBP_1.1C-WT (Fig. 1, A and B). However, with truncations of 160 (MBP_1.1CC160) or 170 (MBP_1.1CC170) residues, 10 µM TNP-ATP fluorescence significantly increased to levels greater than that for MBP_1.1C-WT (Fig. 1). However, truncation of 180 residues (MBP_1.1CC180) dramatically reduced TNP-ATP fluorescence to below that for MBP_1.1C-WT. Because protein production was greatly reduced for MBP_1.1CC180, further study of this construct was not performed. Finally, denaturing each of the MBP fusion proteins with 4 M urea significantly reduced the concentration-dependent TNP-ATP fluorescence increases (data not shown), consistent with TNP-ATP binding as described previously by us (19).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   The effects of truncations of the distal COOH terminus of Kir1.1 on concentration-dependent increases in TNP-ATP fluorescence (Fobs). MBP fusion protein constructs with truncations of 84 (MBP_1.1CC84), 102 (MBP_1.1CC102), 119 (MBP_1.1CC119), 136 (MBP_1.1CC136), 160 (MBP_1.1CC160), 170 (MBP_1.1CC170), and 180 (MBP_1.1CC180) amino acid residues in the COOH terminus of Kir1.1 were produced in bacteria, purified, and 5-µM concentrations were exposed to varying concentrations of TNP-ATP. A, TNP-ATP fluorescence enhancement was monitored with increasing TNP-ATP concentrations. The dashed line represents TNP-ATP increases with full-length COOH terminus (MBP_1.1C-WT) for comparison. TNP-ATP fluorescence increased in a non-linear manner and was well fit by Equation 1 for each construct (solid lines), consistent with TNP-ATP binding. Binding fluorescence increases were greatest for MBP_1.1CC170. The small Fobs with TNP-ATP in 50 mM Tris buffer indicates unbound fluorescence (clear diamonds). B, comparison of maximal 10 µM TNP-ATP Fobs. Number above bar is the number of observations; *, p < 0.01 compared with MBP_1.1C-WT.

Fluorescence enhancement () provides an index to the protein environment provided by the TNP-ATP binding pocket and generally correlates with binding affinity.  values (Fig. 2C) for MBP_1.1CC84, MBP_1.1CC160, and MBP_1.1CC170 were calculated from the protein titration of 5 µM TNP-ATP according to Equation 3. As expected from the comparison of 10 µM TNP-ATP fluorescence values shown in Fig. 1B,  was significantly increased for all three constructs compared with MBP_1.1C-WT (Fig. 2C). The  values for MBP_1.1CC84, MBP_1.1CC160, and MBP_1.1CC170 were 19.3 ± 0.8, 18.7 ± 0.6, and 19.2 ± 0.2, respectively. The value of  used in Equation 1 for the other constructs was that of the MBP_1.1C-WT construct (7.7) because F_obs values were similar (Fig. 1B). The data in Fig. 1A were well fit by Equation 1 (r² > 0.99; p < 0.001).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   The summary of dissociation constant (Kd, µM, (A)), stoichiometry (No (B)), and enhancement factor (; (C)) for TNP-ATP binding to the MBP fusion proteins containing full-length (WT) or truncation constructs with deletion of the terminal 84 (C84), 160 (C160), and 170 (C170) amino acid residues. Kd and No were calculated using Equation 1, using  values derived according to Equation 3 from the protein titration of 5 µM TNP-ATP. *, p < 0.01 compared with MBP_1.1C-WT or MBP_1.1CC84.

TNP-ATP binding affinities (K_d values) and stoichiometries (N) were calculated from Equation 1 for the three constructs that gave 10 µM F_obs values greater than WT (Fig. 1B) and are summarized in Fig. 2. The K_d (µM) for TNP-ATP binding was significantly increased in MBP_1.1CC84 and reduced in MBP_1.1CC160 and MBP_1.1CC170 compared with MBP_1.1C-WT (Fig. 2A): MBP_1.1C-WT = 2.51 ± 0.40; MBP_1.1CC84 = 3.58 ± 0.23; MBP_1.1C C160 = 1.18 ± 0.08; MBP_1.1C C170 = 1.27 ± 0.06. The binding stoichiometry (N_o; TNP-ATP per protein) was reduced from 0.78 ± 0.07 in MBP_1.1C-WT to ~0.5 in the three truncations (Fig. 2B): MBP_1.1CC84 = 0.49 ± 0.02; MBP_1.1C C160 = 0.42 ± 0.02; MBP_1.1C C170 = 0.50 ± 0.02. When taken together, the data in Figs. 1 and 2 demonstrate that as few as 39 amino acid residues in the proximal COOH terminus of Kir1.1 (MBP_1.1C170) are sufficient to bind TNP-ATP.

We also assessed the effects of truncations of the NH₂-terminal end of the COOH terminus of Kir1.1 on the concentration dependence of TNP-ATP fluorescence (Fig. 3). These deletions were from the initial region of the COOH terminus just distal to the second membrane-spanning segment. Recombinant MBP proteins with deletions from 10-124 amino acid residues (MBP_1.1CN10, MBP_1.1CN25, MBP_1.1N50, and MBP_1.1CN124) that lack the first 10, 25, 50, and 124 amino acids of the COOH terminus, respectively, gave no significant TNP-ATP fluorescence increases (Fig. 3, A and B). The MBP_1.1N124 construct consists of the 84 amino acid residues deleted in the MBP_1.1C84 construct shown in Fig. 1A. Thus, deletion of as few as 10 amino acid residues from the NH₂-teminal end of the COOH terminus eliminated TNP-ATP binding.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   The effects of graded truncations of the proximal region of the COOH terminus of Kir1.1 on concentration-dependent increases in TNP-ATP Fobs. MBP fusion protein constructs with truncations of 10 (MBP_1.1CN10), 25 (MBP_1.1CN25), 39 (MBP_1.1CN39), 50 (MBP_1.1CN50), 124 (MBP_1.1CN124) amino acid residues from the initial COOH terminus of Kir1.1 were produced in bacteria, purified, and 5-µM concentrations were exposed to varying concentrations of TNP-ATP. A, TNP-ATP Fobs was monitored with increasing TNP-ATP concentrations. The dashed line (FB) represents free TNP-ATP fluorescence in buffer without protein for comparison. Fobs increased only minimally over that for buffer for any of the truncations, indicating little-to-no significant binding of TNP-ATP to these constructs. B, TNP-ATP binds to the initial 124 (MBP_1.1CC84) but not to the last 84 (MBP_1.1CN124) amino acid residues. C, concentration-dependent TNP-ATP Fobs with deletion of the initial 5 (MBP_1.1CC160(N5)) or 10 (MBP_1.1CC160(N10)) amino acid residues in the MBP_1.1CC160 construct containing the initial 49 amino acids of the Kir1.1 COOH terminus. Fobs was markedly reduced for either the N5 or N10 deletion construct compared with MBP_1.1CC160, indicating near abolishment of TNP-ATP binding. D, TNP-ATP binds to the initial 39 (MBP_1.1CC170) but not to the last 170 (MBP_1.1CN39) amino acid residues. The solid and dashed lines were fit according to Equation 1.

We further examined TNP-ATP fluorescence increases with truncation of the NH₂-terminal end of the MBP_1.1C160 construct that binds TNP-ATP with high affinity (Figs. 1A and 2A). The concentration-dependent TNP-ATP fluorescence increases were virtually abolished with deletion of the first 5 or 10 amino acids in MBP_1.1CC160, MBP_1.1CC160(N5), or MBP_1.1CC160(N10), respectively (Fig. 3C). Thus, the initial 5-10 amino acid residues of the COOH terminus of Kir1.1 are crucial for TNP-ATP binding in the entire COOH terminus as well as the 49-amino acid binding segment in MBP_1.1C160.

To verify that the TNP-ATP binding domain is contained within the initial 39 amino acid residues of the COOH terminus of Kir1.1 (MBP_1.1CC170), we assessed TNP-ATP fluorescence increases with MBP_1.1CC170 compared with MBP_1.1CN39 (Fig. 3D). The latter construct represents the 170-amino acid segment of the COOH terminus following the initial 39 residues. As shown in Fig. 3D, TNP-ATP fluorescence increased significantly in a concentration-dependent manner with MBP_1.1CC170 but not with MBP_1.1CN39. Thus, the initial 39 amino acid residues of the Kir1.1 COOH terminus are sufficient to bind TNP-ATP with high affinity and contain the only TNP-ATP binding site in the Kir1.1 COOH terminus.

The Kinetics of TNP-ATP Binding to the Truncated COOH Terminus of Kir1.1-- The  derived from Equation 3 was used to calculate the protein-bound and free TNP-ATP concentrations according to Equation 4 as described in Ref. 19. Plots of these data for MBP_1.1C160 and MBP_1.1C170 are shown in Fig. 4, A and C, respectively. The data were generally well fit (r² = 0.97 (Fig. 4A) and r² = 0.95 (Fig. 4B); p < 0.01) by Equation 5 for a single binding-site model, although some points did deviate slightly from the curve. The reason for the slight deviations is not clear, although this may involve some cooperativity associated with protein-protein interactions in the multimeric forms of these proteins shown previously (1). K_d values were similar to those determined from the F_obs data in Fig. 1, and B_max gave an N₀ (1.9 µM TNP-ATP/5 µM protein) of 0.38 mol of TNP-ATP/mol protein for MBP_1.1CC160 and (2.4 µM TNP-ATP/5 µM protein) of 0.48 mol of TNP-ATP/mol protein for MBP_1.1CC170. Scatchard plots for MBP_1.1C160 and MBP_1.1C170 are shown in Fig. 4, B and D, respectively. The Scatchard data were well fit by a linear non-competitive binding equation (19) as described by Moczydlowski and Fortes (26) and yielded kinetic parameters similar to those calculated from the bound versus free curves.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Kinetics of TNP-ATP binding to MBP_1.1CC160 and MBP_1.1CC170. A and C, 5-µM protein concentrations were exposed to varying concentrations of TNP-ATP, and bound and free concentrations of TNP-ATP were calculated by Equation 4 using the enhancement factors calculated from Equation 3. The curves were fit according to Equation 5. Maximal TNP-ATP bound (Bmax) values are shown. B and D, Scatchard plots of TNP-ATP binding to MBP_1.1C160 and MBP_1.1C170. The Scatchard data were well fit by a linear non-competitive binding equation (19) as described by Moczydlowski and Fortes (26). The TNP-ATP/protein stoichiometries N (nmol/mg) and No (Equation 1; mol/mol) and the Kd (mM) derived from the Scatchard analyses are shown.

Mutation of Single Residues in the First 39 Amino Acids of the COOH Terminus Abolish TNP-ATP Binding-- Mutation of a number of residues in the COOH terminus of Kir6.2 have been shown to reduce sensitivity of this K_ATP channel to ATP and/or PIP₂ (14, 27-29). Because channel gating is a complex process involving at a minimum both the binding of ATP and the subsequent closing of the channel pore, we examined the effects of some of these mutations on TNP-ATP binding to MBP_1.1CC170. We focused on four arginine residues, Arg¹⁸⁸, Arg²⁰³, Arg²¹², and Arg²¹⁷, in Kir1.1 (ROMK1) that are conserved in Kir6.x COOH termini (Fig. 5) and have been shown to alter Kir6.2 channel gating by ATP or PIP₂. The effects of mutation of these residues in MBP_1.1CC170 on the concentration dependence of TNP-ATP F_obs are shown in Fig. 6. Mutation of Arg¹⁸⁸ to either a neutral residue, MBP_1.1CC170(R188A/Q), or to a negatively charged residue, MBP_1.1CC170(R188E), resulted in a similar loss of TNP-ATP-dependent increases in F_obs (Fig. 6A). Thus, for the other conserved arginine residues we assessed only the Arg to Ala mutations. TNP-ATP binding was markedly reduced in the MBP_1.1CC170(R203A) and MBP_1.1CC170(R217A) mutant constructs (Fig. 6, B and D, respectively). In contrast, mutation of Arg²¹², MBP_1.1CC170(R212A), had no significant effect on the concentration-dependence of TNP-ATP increases in F_obs compared with MBP_1.1CC170 (Fig. 6C). The  for MBP_1.1CC170(R212A) was significantly higher, whereas the K_d was significantly lower than for MBP_1.1CC170: () 30.2 ± 0.7 versus 19.2 ± 0.2 (p < 0.01); (K_d) 0.60 ± 0.09 versus 1.27 ± 0.06. 

View larger version (22K): [in this window] [in a new window]   Fig. 5.   A, summary of arginine mutations in the first 39-amino acid residue segment, MBP_1.1CC170, of Kir1.1 (ROMK1). The mutated arginine residues are indicated by * with the ROMK1 residue number indicated. The numbers in parentheses are the corresponding conserved residues in rat Kir6.2. B, alignment of the initial COOH-terminal segments of KATP channels. Identical/conserved residues are shaded. Single letter amino acid codes are used.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   TNP-ATP binding to the MBP fusion proteins with mutations of single conserved arginine residues in MBP_1.1CC170. The 39-amino acid region of MBP_1.1CC170 with the specific arginine mutation is shown at the top of each panel. Concentration-dependent increases in TNP-ATP Fobs are plotted for each construct with lines fit using Equation 1. The wild-type (WT) MBP_1.1CC170 is shown for comparison (clear squares, dashed line). The low basal fluorescence of free TNP-ATP in buffer is also shown for comparison (clear diamonds). The effects of denaturing proteins with 4 M urea (clear circles, B, and solid triangles, C and D) are shown in panels B-D. Mutations of arginine 188 to alanine (MBP_1.1CC170(R188A)), arginine 203 to alanine (MBP_1.1CC170(R203A)), and arginine 217 to alanine (MBP_1.1CC170(R188A)) significantly reduced TNP-ATP fluorescence increases due to binding. Mutation of arginine 212 to alanine (MBP_1.1CC170(R212A)) had no significant effect on TNP-ATP fluorescence increases, i.e. TNP-ATP binding.

PIP₂ Competition of TNP-ATP Binding to the Truncated COOH Terminus of Kir1.1 MBP Fusion Proteins-- Our recent observations (2) indicated that PIP₂ directly bound to the COOH termini of Kir1.1 and that PIP₂ competed with TNP-ATP for the binding to MBP_1.1C. Here we further evaluated the ability of PIP₂ to compete for TNP-ATP binding to the COOH-terminal 39-amino acid binding segment of Kir1.1. Fig. 7A (left panel) shows the typical TNP-ATP fluorescence increases associated with binding of this nucleotide to MBP_1.1C170. Following binding of 10 µM TNP-ATP, PIP₂ was added in increasing concentrations (Fig. 7A, right panel). PIP₂ reduced 10 µM TNP-ATP F_obs in MBP_1.1CC170 in a concentration-dependent manner with an EC₅₀ of 10.9 ± 0.2 µM. Because TNP-ATP binding was unaltered in the MBP_1.1C170(R212A) mutant construct (Fig. 6C) and mutation of a similar arginine residue to the Kir6.2 COOH terminus reduced PIP₂ gating sensitivity (14), we assessed the ability of PIP₂ to compete TNP-ATP binding in MBP_C170(R212A). Using a similar protocol to that shown in Fig. 7A, Fig. 7B shows that PIP₂ reduced TNP-ATP binding to MBP_1.1C170(R212A) with an EC₅₀ of 5.7 ± 0.7 µM (p < 0.001 compared with MBP_1.1C170). Thus, the R212A mutation enhanced, rather than diminished, the ability of PIP₂ to compete TNP-ATP binding to MBP_1.1CC170.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   PIP2 reduces TNP-ATP binding to MBP fusion proteins containing the first 39 amino acid residues of Kir1.1 COOH terminus (MBP_1.1CC170) (A) or MBP_1.1CC170 with arginine 212 mutated to alanine (MBP_1.1CC170(R212A) (B). A and B, (left panels) concentration-dependent increases in TNP-ATP fluorescence (solid squares). Solid lines fit using Equation 1. A and B, (right panels) PIP2 decreases TNP-ATP fluorescence in a dose-dependent manner to the levels seen with buffer alone (open squares). The solid line was fit to a downhill dose-dependent model (GraphPad Prism v. 3.02). EC50 values for PIP2 competition of TNP-ATP binding are shown in each panel.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our previous study provided direct evidence that TNP-ATP can bind to the COOH termini of Kir1.1 and the ATP-sensitive K⁺ channels, Kir6.1 and Kir6.2 (19). Because TNP-ATP did not bind to the NH₂ termini of Kir1.1 or Kir6.1, the COOH terminus of ATP-regulated K⁺ channels was both necessary and sufficient to bind nucleotides. The present observations support the suggestion that ATP inhibition of K_ATP channel activity is mediated by interaction of ATP with the COOH terminus of the Kir subunit. Here we have further localized the nucleotide binding segment of Kir1.1 to the initial 39 amino acid residues of the COOH terminus. In addition, we show that specific residues within this segment are critical to nucleotide binding. Finally, we demonstrate that this 39-residue segment also retains the critical sequences necessary for PIP₂ competition of TNP-ATP binding.

Several lines of evidence indicate that the initial 39 amino acid residues of the COOH terminus of Kir1.1 is the minimal segment necessary for the high affinity binding of TNP-ATP and for PIP₂ competition and that this segment is the only significant adenine nucleotide-binding region along the COOH terminus of Kir1.1. First, MBP_1.1CC170 bound TNP-ATP with higher affinity than the entire COOH terminus (Figs. 1 and 2, MBP_1.1C-WT) or any of the other truncation constructs (Figs. 1-4). In addition, TNP-ATP binding to the construct with a 10-residue greater truncation (MBP_1.1CC180) was dramatically reduced compared with MBP_1.1CC170 (Fig. 1). Moreover, MBP_1.1C170 exhibited a significantly higher  (Fig. 2C) than for MBP_1.1C-WT, consistent with an improved protein environment for TNP-ATP binding. Second, the 170-amino acid segment of the COOH terminus beyond the initial 39 amino acid residues (MBP_1.1CN39) did not exhibit significant TNP-ATP binding (Fig. 3D), demonstrating that nucleotide binding is limited to the first 39 amino acids of the COOH terminus. Third, truncations of as few as 5 amino acids from the NH₂-terminal end of the COOH terminus (Fig. 3C) virtually abolished TNP-ATP binding. Fourth, mutation of any one of three individual residues (Fig. 6, Arg¹⁸⁸, Arg²⁰³, or Arg²¹⁷) within the 39-amino acid MBP_1.1CC170 construct abolished TNP-ATP binding, indicating that they are directly involved in the formation of the TNP-ATP binding structure of Kir1.1. The observation that R212A did not alter TNP-ATP binding (Fig. 6C) indicates that the effects of R188A (Fig. 6A), R203A (Fig. 6B), and R217A (Fig. 6D) on TNP-ATP binding are unlikely to be nonspecific.

All three arginine residues that affect TNP-ATP binding in Kir1.1 (Arg¹⁸⁸, Arg²⁰³, and Arg²¹⁷) are conserved in Kir6.x channels (Fig. 5) as well as most Kir channels, the only exception being that Arg²⁰³ is a histidine residue in Kir4.1. Mutation of any of these three residues in Kir6.2 (Arg¹⁷⁷, Arg¹⁹², or Arg²⁰⁶; Fig. 5) has been shown to alter channel gating by ATP and/or PIP₂ (14). Because ATP and PIP₂ are competitive regulators of K_ATP channel gating and we have shown that TNP-ATP and PIP₂ interactions occur within the same 39-amino acid segment (Fig. 7), this mutational study in Kir6.2 is fully consistent with our observations of the critical roles of Arg¹⁸⁸ and Arg²¹⁷ in nucleotide binding (Fig. 6). Interestingly, the R201A mutation in Kir6.2 (Fig. 5A), corresponding to R212A in Kir1.1, reduces Kir6.2 channel gating by ATP and PIP₂, whereas this mutation in Kir1.1 has no effect on TNP-ATP binding (Fig. 6C) and enhances competition of TNP-ATP binding by PIP₂ (Fig. 7B). This suggests that Arg²⁰¹ in Kir6.2 functions differently than Arg²¹² in Kir1.1 for ATP binding or that Arg²⁰¹ in Kir6.2 affects channel gating by a mechanism other than modification of direct binding of ATP. Finally, a number of other residues in the first 39 amino acids of Kir6.2 have also been shown to affect ATP and/or PIP₂ gating of channel activity (14, 27-29) or the binding of 8-azido-[³²P]ATP (30), consistent with the role of the initial region of the Kir6.2 COOH terminus, and by analogy the Kir1.1 COOH terminus, in ATP/PIP₂ effects.

We previously suggested that TNP-ATP binds to the COOH termini of Kir1.1 and the Kir6.x ATP-sensitive channels with a stoichiometry of at least one molecule of ATP binding per COOH-terminal protein (19). The TNP-ATP/protein stoichiometry of 0.78 for the entire Kir1.1 COOH terminus (MBP_1.1C-WT) confirms our previous observations of a single adenine nucleotide binding site for each of the four Kir subunits forming the kidney ATP-regulated K⁺ channel. The present results provide additional support for this model by demonstrating that TNP-ATP binding is confined to a single short segment in the COOH terminus of Kir1.1. Although the N_o for MBP_1.1CC170 (Fig. 2B) was reduced compared with WT, the stoichiometry of 0.5 is still consistent with one binding site per protein molecule. The reduced stoichiometry was also observed in MBP_1.1CC84 and MBP_1.1CC160, suggesting that amino acids in the distal COOH terminus may be involved in the stabilization of the proximal 39-residue adenine nucleotide binding pocket.

One of the major regulatory mechanisms of Kir1.1 and K_ATP channels, as well as other Kir K⁺ channels, is channel gating (opening) by phosphatidylinositol phospholipids like PIP₂. PIP₂ competition of the inhibitory action of ATP provides a mechanism for opening K_ATP channels in intact cells in the presence of millimolar concentrations of cytosolic ATP. We have recently reported that PIP₂ competed with TNP-ATP binding to the COOH terminus of Kir1.1 and Kir6.x channels. Our present results show that the 39-amino acid nucleotide binding domain in the COOH termini of Kir1.1 retains the ability of PIP₂ to compete off TNP-ATP. Thus, the initial 39-amino acid residue segment in the COOH terminus of Kir1.1 represents both the ATP binding domain and a PIP₂ binding segment. The enhancement, rather than diminution, of PIP₂ competition of TNP-ATP binding in the R212A mutant suggests that the effect of mutation of the similar residue in Kir6.2 (14) on PIP₂-ATP interactions may involve a mechanism other than ATP-PIP₂ competition for binding. It should be noted, however, that although the initial COOH-terminal sequences of Kir1.1 and Kir6.2 are similar, they are not identical. Thus, it is possible that certain distinct amino acid residues in Kir6.2 may change the role of this arginine residue in PIP₂ binding compared with Kir1.1.

In summary, we have defined a 39-amino acid segment in the Kir1.1 (ROMK) COOH terminus that forms an ATP-PIP₂ binding domain and have identified three arginine residues that significantly alter TNP-ATP binding. Because this region is similar and the three critical arginine residues are conserved in Kir6.x channels (Fig. 5B), it seems likely that the initial COOH-terminal segment in K_ATP channels also forms an ATP-PIP₂ binding domain.

	FOOTNOTES

^* This work was supported by Grant DK54999 from the National Institutes of Health (to S. C. H.).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.

Both authors contributed equally to this work.

^§ To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., SHM B147, P.O. Box 208026, New Haven, CT 06520-8026. Tel.: 203-785-6696; Fax: 203-785-7678; E-mail: steven.hebert@yale.edu.

Published, JBC Papers in Press, October 14, 2002, DOI 10.1074/jbc.M208679200

	ABBREVIATIONS

The abbreviations used are: PIP₂, phosphatidylinositol 4,5 diphosphate; MBP, maltose binding protein; a.u., arbitrary unit; WT, wild type; TNP, 2',3'-O-(2,4,6-trinitrophenylcyclo-hexadienylidene); ROMK, renal outer medulla potassium channel.

	REFERENCES

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