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J. Biol. Chem., Vol. 277, Issue 26, 23260-23270, June 28, 2002

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The Carboxyl Termini of K_ATP Channels Bind Nucleotides^*

Carlos G. Vanoye^§, Gordon G. MacGregor^§¶, Ke Dong^§¶, LieQi Tang^§¶, Alexandra S. Buschmann^¶, Amy E. Hall^¶, Ming Lu^¶, Gerhard Giebisch^¶, and Steven C. Hebert^¶

From the ^¶ Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026 and the  Department of Medicine, Division of Genetic Medicine, Vanderbilt University Medical School, Nashville, Tennessee 37232-6304

Received for publication, December 17, 2001, and in revised form, April 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ATP-sensitive potassium (K_ATP) channels are expressed in many excitable, as well as epithelial, cells and couple metabolic changes to modulation of cell activity. ATP regulation of K_ATP channel activity may involve direct binding of this nucleotide to the pore-forming inward rectifier (Kir) subunit despite the lack of known nucleotide-binding motifs. To examine this possibility, we assessed the binding of the fluorescent ATP analogue, 2',3'-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)adenosine 5'-triphosphate (TNP-ATP) to maltose-binding fusion proteins of the NH₂- and COOH-terminal cytosolic regions of the three known K_ATP channels (Kir1.1, Kir6.1, and Kir6.2) as well as to the COOH-terminal region of an ATP-insensitive inward rectifier K⁺ channel (Kir2.1). We show direct binding of TNP-ATP to the COOH termini of all three known K_ATP channels but not to the COOH terminus of the ATP-insensitive channel, Kir2.1. TNP-ATP binding was specific for the COOH termini of K_ATP channels because this nucleotide did not bind to the NH₂ termini of Kir1.1 or Kir6.1. The affinities for TNP-ATP binding to K_ATP COOH termini of Kir1.1, Kir6.1, and Kir6.2 were similar. Binding was abolished by denaturing with 4 M urea or SDS and enhanced by reduction in pH. TNP-ATP to protein stoichiometries were similar for all K_ATP COOH-terminal proteins with 1 mol of TNP-ATP binding/mole of protein. Competition of TNP-ATP binding to the Kir1.1 COOH terminus by MgATP was complex with both Mg²⁺ and MgATP effects. Glutaraldehyde cross-linking demonstrated the multimerization potential of these COOH termini, suggesting that these cytosolic segments may directly interact in intact tetrameric channels. Thus, the COOH termini of K_ATP tetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ATP-sensitive or ATP-regulated potassium (K_ATP)¹ channels couple metabolism to either cell excitability (Kir6.x) (1-6) or potassium secretion (Kir1.1 in kidney) (7, 8) and provide therapeutic targets for diseases including tissue ischemia, diabetes, hypertension, and disorders of potassium homeostasis. K_ATP channels are formed by an octameric complex of four pore-forming subunits (Kir6.x or Kir1.1) and four sulfonylurea receptors, SUR1 or SUR2 for Kir6.x (2) or the cystic fibrosis transmembrane conductance regulator or SUR2b for Kir1.1 (9, 10).

Although the SUR/fibrosis transmembrane conductance regulator subunits contain nucleotide-binding folds (11, 12), this subunit is not required for ATP-mediated inhibition of K⁺ channel activity. For example, deletion of the last 36 amino acids from the COOH terminus of Kir6.2 (Kir6.2C36) produces functional K⁺ channels in the absence of coexpressed SURs that are sensitive to ATP (13). Nevertheless, SUR subunits are required for ADP-mediated activation of K_ATP channels (14-16). Thus, ATP inhibition of K_ATP channel activity is thought to involve direct interaction with Kir subunits despite the lack of identifiable nucleotide-binding motifs. The recent demonstration of the photoaffinity labeling of Kir6.2 channel by 8-azido-[-³²P]ATP (17, 18) also supports the direct binding of ATP with the pore-forming subunit of K_ATP channels. In addition, mutations in both the NH₂- and COOH-terminal regions of the Kir6.2 (13, 19-23) and Kir1.1 (24) subunits alter the EC₅₀ for ATP-mediated channel gating. Because ATP-mediated inhibition of channel activity must be a complex process involving residues that form an ATP-binding pocket and others that may be required for linking ATP binding to channel closure, those mutational studies of channel gating by nucleotides do not provide unequivocal evidence for direct involvement of those residues in ATP binding.

In the present study, we assessed the direct binding of fluorescent 2',3'-O-(2,4,6-trinitrophenylcyclo-hexadienylidene) adenosine triphosphate (TNP-ATP) to purified maltose-binding fusion proteins of the cytosolic NH₂ and COOH termini of the three known K_ATP channels and the COOH terminus of a ATP-insensitive inward rectifier K⁺ channel, Kir2.1 (25). We provide herein what we believe to be the first evidence of direct binding of ATP to cytosolic domains of the pore-forming subunits of K_ATP channels and show that the COOH termini, but not the NH₂ termini, of Kir subunits of K_ATP channels bind TNP-ATP. The kinetic analyses of TNP-ATP binding suggest that the COOH termini have a single nucleotide-binding site. Based on glutaraldehyde cross-linking studies, the COOH termini of these three ATP-sensitive channels also exhibit multimerization potential so that they may interact in these intact tetrameric channels.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

DNA Constructs and Mutagenesis-- DNAs encoding the NH₂ and COOH termini of Kir6.1 (encoding amino acids 1-73 and 178-424 (247 amino acids), respectively) and the COOH terminus of Kir6.236 (encoding amino acids 169-354 (186 amino acids)) were obtained by reverse transcription-PCR from rat kidney and brain, respectively. The NH₂ and COOH termini of Kir1.1 (encoding amino acids 1-80 and 183-391 in ROMK2 (209 amino acids), respectively) were derived from the previously cloned rat Kir1.1 (26, 27). The COOH terminus of mouse Kir2.1 encoded amino acids 179-428 (250 amino acids). The sequences of all of the constructs were confirmed using the cycle sequencing method (Keck Facility, Yale). All channel cDNA constructs were ligated into the pMBPT vector kindly provided by Dr. G. A. Altenberg (28). The vector was derived from the MAL^TM-c2 vector (maltose-binding protein (MBP) fusion vector; New England Biolabs).

Production and Purification of Maltose-binding Fusion Proteins-- We constructed MBP fusion proteins containing the NH₂ (MBP_1.1N and MBP_6.1N) or the COOH (MBP_1.1C and MBP_6.1C) terminus of rat Kir1.1 and Kir6.1, respectively, and the COOH termini of mouse Kir2.1 (MBP_2.1C) and rat Kir6.2C36 (MBP_6.2C36) channels. We used the MBP_6.2C36 construct for these studies because deletion of the last 36 amino acids from the end of the COOH terminus of Kir6.2 gives rise to functional and ATP-sensitive channel activity in cells in the absence of SUR1 (13). Recombinant proteins were expressed using the pMBPT vector as per the manufacturer's instructions (New England Biolabs). Briefly, 1 liter of Luria-Bertani medium with 0.1 mg/ml ampicillin and 0.5% glucose was inoculated with 10 ml of an overnight culture of Epicurian coli® BL21-CodonPlus^TM-RIL-competent cells (Stratagene) 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 2.5 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 (20 mM Tris-Cl, 200 mM NaCl, 1 mM EDTA, pH 7.4) and frozen overnight at 20 °C. The sample was thawed in ice water and lysed with a probe sonicator (four times for 30 s, with 30-s intervals in an ice water bath. The sample was then centrifuged at 9,000 × g for 30 m 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 with 10 mM maltose, and 1.5-ml fractions were collected. The protein was detected by UV absorbance at 280 nm, dialyzed against 50 mM Tris-HCl, pH 7.5, and kept at 80 °C until the experiments were performed. The yields of purified recombinant fusion proteins were 15-25 mg/liter.

TNP-ATP Binding-- To assess the binding of ATP to these recombinant fusion proteins, we used fluorescent TNP-ATP (Molecular Probes, Inc.) (29, 30), which has been widely employed to study nucleotide binding to enzymes and other proteins (31-34). The binding of TNP-ATP to recombinant proteins was performed generally as described by Faller (32). Briefly, 5 µM recombinant protein was dissolved in 50 mM Tris-Cl at pH 7.5 or 5 mM MES monohydrate (Sigma) at pH 6.5, and TNP-ATP binding was detected by the increase in fluorescence upon binding to recombinant protein using a SPEX Fluromax-3 spectrofluorometer (Jobin Yvon Inc., Edison, NJ). The fluorescence units reported here were scaled by 1000. Excitation wavelength (403 nm) and emission wavelength (546 nm) were determined for the Kir1.1 COOH terminus fusion protein and utilized for all recombinant proteins (slit widths, 5 nm) because they did not vary significantly among proteins examined. A typical 10-nm blue shift in emission wavelength was detected upon binding of TNP-ATP to proteins (32). The temperature was maintained at 22 ± 0.1 °C by a circulating water bath (Neslab, Newington, NH). Incremental additions of TNP-ATP were delivered to polystyrene cuvettes (Elkay Products Inc., Shrewsbury, MA) from stock solutions (0.2-1.0 mM). Total fluorescence was measured 30 s after the additions to allow for equilibration. All of the titrations were corrected for dilution. TNP-ATP fluorescence was also measured in the presence of 5 mM MgATP or by denaturing the protein with 4 M urea. MgATP was added from a stock solution of 0.2 M adjusted to pH 7.5 or 6.5, as indicated.

Free TNP-ATP is weakly fluorescent in buffer, but upon binding to proteins fluorescence is enhanced severalfold with the absolute magnitude dependent on the specific protein environment within the nucleotide-binding pocket (31, 32). The fluorescence enhancement factor (), TNP-ATP to protein subunit stoichiometry (N_o), and dissociation constant (K_d (µM)) were determined by least squares fitting to a modified version of the binding equation derived by Faller (32) using GraphPad PRISM^TM 3.0 software. The observed fluorescence intensity (F_obs) in arbitrary units is given by the following equation.

(Eq. 1)

where P is the protein concentration (µM). Q and Q₂ are constants (fluorescence intensity/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 (32): 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.

(Eq. 2)

Concentrations of TNP-ATP above 20 µM were not used to minimize inner filter effects. Protein light scatter intensity (F_{light scatter}) was subtracted from all F_obs values. The concentration dependence of light scattering of individual recombinant proteins was: F_{light scatter} = RP + R₂P², where R and R₂ are constants (light intensity/µM and µM² of protein, respectively).

We independently determined the enhanced factor () by measuring the increase in F_obs with increasing protein concentration at a fixed concentration of TNP-ATP (5 µM). The F_obs data were corrected for light scatter and were fit well by a single exponential. F was determined as F_obs at infinite protein concentration when all TNP-ATP would be bound. The enhancement factor was then calculated as follows.

(Eq. 3)

Using this enhancement factor we calculated the concentrations of free ([F]) and bound ([B]) TNP-ATP as described by Moczydlowski and Fortes (31) taking into account the inner filter effect.

(Eq. 4)

Free TNP-ATP 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.

(Eq. 5)

where B_max is the maximal TNP-ATP binding. The data were also plotted for Scatchard or Hill analyses (36) as described (31, 37, 38). For noncompetitive binding the Scatchard analysis is linear as described by Moczydlowski and Fortes (31).

(Eq. 6)

where N is the number of TNP-ATP binding sites in µmol/mg.

For MgATP, NaATP, or MgCl₂ competition of TNP-ATP binding, we used a two-site model as described by Faller (39).

(Eq. 7)

where F_obs/F is the fractional change in fluorescence intensity, S_frac is the fraction of binding sites in the first site, and K₁ and K₂ are the apparent substrate affinities for the first and second sites, respectively.

8-Azido-[-³²P]ATP Labeling-- Photoaffinity labeling of recombinant proteins with 8-azido-[-³²P]ATP was performed as described previously (40, 41). 5 µg of the purified protein was added to solution A (50 mM HEPES, 10 mM Tris, pH 7.4, 10 mM CaCl₂, 0.5 mM MgCl₂, and 2 µCi of [-³²P]azido-ATP; ICN Biochemicals, Inc.) and incubated for 15 min in the dark at 4 °C. The reaction mixture was irradiated with UV light at 350 nm for 1 min at room temperature to covalently link the azido-ATP to neighboring amino acid residues. The labeled protein was resolved by SDS-PAGE and visualized by autoradiography.

Cross-linking-- Cross-linking of fusion proteins with glutaraldehyde was performed as described previously (42). Briefly, 0.15 µg of purified MBP fusion proteins (total volume, 40 µl) were incubated with different concentrations (final concentrations, 0, 0.005, 0.01, 0.025, 0.05, 0.075, and 0.1%) of glutaraldehyde in phosphate-buffered saline on ice for 30 min. The cross-linking was quenched with the addition of 100 mM glycine, pH 8.0. The proteins were solubilized in Laemmli buffer with 5% -ME and resolved by SDS-7.5% PAGE. The proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad), blocked with 5% milk in a shaker at room temperature for 1 h, incubated with rabbit anti-MBP antibody (1:10,000; New England Biolabs) overnight at 4 °C on a rocker, and then incubated with horseradish peroxidase-conjugated donkey anti-rabbit Ig (1:10,000; Amersham Biosciences) for 1 h at room temperature on a rocker. The proteins were visualized by ECL (Amersham Biosciences).

Electrophysiology-- Inside out patch-clamp experiments were performed at room temperature (22-24 °C) as described (V_p = 40 mV) (43) to assess the effects of TNP-ATP on apical K_ATP channel activity in rat cortical collecting ducts principal cells. Briefly, Sprague-Dawley rats (80-100g) were obtained from Taconic Farms Inc. and kept on normal chow diet (PMI Nutrition International, Inc.) for 7-10 days before experiments. The animals were euthanized, their kidneys were removed, and coronary slices were cut and placed in ice-cold dissection solution. Individual cortical collecting ducts were dissected at room temperature, and the tubules were immobilized on a 5 × 5-mm cover glass coated with Cell Tac (Becton Dickinson) and then transferred to a perfusion chamber mounted on the stage of an inverted microscope (IMT-2; Olympus). The tubules were opened with a sharpened pipette to gain access to the apical membrane. The principal cells were identified by their hexagonal shape and large flat surface. The bath solution contained 140 mM NaCl, 5 mM KCl, 1 mM EGTA, 10 mM HEPES, 0.2 mM MgATP, pH 7.4. The pipette solution contained 140 mM KCl, 1.8 mM MgCl₂, 10 mM HEPES, pH 7.4. TNP-ATP (0-1000 µM) was added to the bath solution where indicated. MgATP is required in the bath solution to keep the K_ATP channels in principal cells from running down (43).

Chemicals-- All of the chemicals were research grade or better and were from Sigma unless otherwise stated.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ATP Binds to the COOH Terminus of Kir1.1-- All MBP fusion proteins were efficiently expressed in bacteria and could be highly purified at milligram quantities (5-25 mg/liter of bacterial culture) without exposure to detergents or denaturing agents (28). The recombinant MBP and the NH₂-terminal (MBP_1.1N and MBP_6.1N) and COOH-terminal (MBP_1.1C, MBP_6.1C, MBP_6.2C36, and MBP_2.1C) MBP fusion proteins ran at their expected molecular masses as shown in Fig. 1. MBP_6.2C36 consistently produced the lowest yield of 5-10 mg/liter, whereas the yields of MBP_1.1C and MBP_6.1C were 15-25 mg/liter. Cleaving the MBP from the channel protein at the thrombin site resulted in insoluble protein under our current buffer conditions, probably because of the hydrophobicity of these cytosolic NH₂ and COOH termini. Thus, all of the experiments were performed using the MBP fusion proteins.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Expression of MBP fusion proteins. MBP proteins containing the NH2 (designated by N) and COOH termini (designated by C) of rat Kir1.1, Kir6.1, Kir6.2C36, and Kir2.1 channels were efficiently expressed in bacteria as soluble proteins in the absence of detergents. Separation of purified MBP or MBP fusion proteins of MBP_2.1C, MBP_1.1N, and MBP_1.1C (A) or MBP_6.1N, MBP_6.1C, and MBP_6.2C36 (B) on 10% SDS-PAGE in the presence of reducing agents yields bands of the appropriate sized products. The bands were visualized by Coomassie Brilliant Blue staining.

We used fluorescent TNP-ATP to assess the binding of ATP to the cytosolic domains of Kir channels (31-34). The concentration dependence relationships of TNP-ATP fluorescence with MBP_1.1C, MBP_1.1N, and MBP alone at pH 7.5 are shown in Fig. 2. F_obs for unbound TNP-ATP in buffer without protein was low and increased in a nonlinear, concentration-dependent manner (Fig. 2, A and B), consistent with the intrinsic fluorescence of this ATP analogue and the inner filter effect (29, 30, 31). All of the buffer data were well fit using a second order polynomial that accounts for this inner filter effect (see "Materials and Methods"; r²  0.99). In contrast, F_obs was significantly enhanced over the buffer control in the presence of MBP_1.1C (Fig. 2, A and B, F_P), consistent with binding of TNP-ATP to this fusion protein. F_obs with MBP_1.1C saturated (Fig. 2B) and was well fit by Equation 1 (r² = 0.999) using a  of 7.7 (see Fig. 5) and gave a K_d of 2.64 ± 0.26 µM (n = 11). Denaturing MBP_1.1C protein with 4 M urea (Fig. 2A, F_P Urea; n = 15) or 0.1% SDS (Fig. 2B, F_P SDS; n = 7) diminished the nucleotide concentration-dependent increase in F_obs to values close to that of TNP-ATP in the urea or SDS buffers without protein, respectively. The increase in F_obs with MBP_1.1C was not due to TNP-ATP interactions with MBP because the TNP-ATP concentration-dependent increase in F_obs with MBP (Fig. 2C, F_P; n = 5) was similar to the TNP-ATP curve in buffer alone (Fig. 2A, buffer) and was not significantly different in the absence or presence of 5 mM MgATP (Fig 2C; F_P MgATP) or 4 M urea (Fig. 2C; F_P Urea). The binding of TNP-ATP was specific for the COOH terminus of Kir1.1 because the increase in F_obs with MBP_1.1N was small and unaffected by 5 mM MgATP or 4 M urea (Fig. 2D; n = 10). Mixing of MBP_1.1N and MBP_1.1C (1:1) did not significantly affect the affinity for TNP-ATP binding (control K_d = 1.84 ± 0.14, (n = 6); mixing K_d = 1.63 ± 0.22; (n = 5); data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   TNP-ATP binds to the MBP fusion protein containing the COOH terminus of Kir1.1 (MBP_1.1C), but not to the NH2-terminal fusion protein (MBP_1.1N) or to MBP without a fusion construct. All of the studies were performed using 5 µM protein concentrations in 50 mM Tris buffer at pH 7.5. A and B, the TNP-ATP concentration-dependent increases in total observed fluorescence, Fobs, (solid squares; FP; solid line calculated according to Equation 1; A and B) and Fobs in the presence of 4 M urea (FP  Urea, solid triangle and solid line; A) or 0.1% SDS (FP SDS, white squares and solid line; B) are shown. Urea and SDS significantly reduced Fobs. Fobs in normal (FB; white squares and dashed line in A; diamonds and dashed line in B) or urea (FB Urea, white triangles and dashed line in A) buffers without protein is shown for comparison. The intersection of linear fitting (B, dashed lines) to the initial and final Fobs data is shown (see text for discussion). C, TNP-ATP concentration-dependent increases in Fobs with MBP without fusion protein are low (FP) and unaffected by 5 mM MgATP (FP MgATP) or 4 M urea (FP Urea), indicating no TNP-ATP binding to MBP. D, TNP-ATP does not bind to MBP_1.1N; Fobs with increasing TNP-ATP is low and unaffected by 5 mM MgATP (FP MgATP) or 4 M urea (FP Urea).

Further support for nucleotide binding to MBP_1.1C was obtained by photoaffinity labeling by 8-azido-[-³²P]ATP as shown in Fig. 3A. The 8-azido-[-³²P]ATP labeling was competed with unlabeled MgATP consistent with specific labeling of MBP_1.1C with this nucleotide analogue. We also examined the ability of MgATP to compete the TNP-ATP binding to MBP_1.1C. The TNP-ATP concentration-dependent increase in F_obs with MBP_1.1C was reduced by 5 mM MgATP (Fig. 3B, triangles), and the K_d for TNP-ATP binding affinity was significantly increased; K_d increased from 3.0 ± 0.2 (F_P) to 6.9 ± 1.9 (F_{P 5 mM MgATP}; n = 13). Increasing MgATP concentration to 50 mM virtually abolished TNP-ATP fluorescence enhancement with MBP_1.1C (K_d = 50.9 ± 14.7 µM; Fig. 3B; n = 5). We also assessed the competition of TNP-ATP binding to MBP_1.1C by MgATP (Fig. 3C). Increasing concentrations of MgATP reduced F_obs/F in a concentration-dependent manner. The shape of the MgATP competition curve was complex, suggesting multiple binding interactions; the data were well fit, however, using the two-site model described by Equation 7 (r² = 0.99). K₁ and K₂ were 71 ± 5 and 3.8 ± 0.8 mM, respectively, and S_frac was 0.77 ± 0.02. 

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Competition of nucleotide binding to the COOH-terminal MBP fusion protein of Kir1.1 (MBP_1.1C). A, MBP_1.1C was photoaffinity labeled by 8-azido-[-32P]ATP in the absence or presence of varying concentrations of MgATP. 8-Azido-[-32P]ATP label MBP_1.1C and labeling is competed by µM concentrations of MgATP. B, MgATP competes TNP-ATP binding to MBP_1.1C. TNP-ATP concentration-dependent increases in Fobs with 5 µM MBP_1.1C in control buffer (FP; squares), or buffer containing either 5 mM (FP 5 mM MgATP; triangles) or 50 mM MgATP (FP 50 mM MgATP; diamonds). 5 mM MgATP reduced Fobs by ~50% at 10 µM TNP-ATP, whereas 50 mM MgATP abolished TNP-ATP fluorescence increases. Fobs with buffer containing 50 mM MgATP without protein is shown for comparison (FB 50 mM MgATP; circles and dashed line). C, competition of TNP-ATP binding to MBP_1.1C by MgATP (squares; line derived from Equation 7), MgCl2 (diamonds; dashed line using one-site component of Equation 7), and NaATP (triangles; dashed line using one-site component of Equation 7). 5 µM MBP_1.1C was loaded with 10 µM TNP-ATP, steady-state maximal fluorescence (F) was measured, and then the relative changes in Fobs (Fobs/F) were measured with the addition of the indicated substances (S). See text for EC50 values. D, 1 mM MgCl2 reduces TNP-ATP binding affinity. TNP-ATP concentration-dependent increases in Fobs with 5 µM MBP_1.1C (FP) was reduced by 1 mM MgCl2 (FP 1 mM MgCl2). Intrinsic TNP-ATP fluorescence in 50 mM Tris-Cl buffer (pH 7.5) with 1 mM MgCl2 is shown for comparison (FB; 1 mM MgCl2).

A fraction of MgATP will dissociate in our buffer solution to free Mg²⁺ and ATP anion (43), and Mg²⁺ has been shown to modulate TNP-ATP binding or fluorescence enhancement in several nucleotide-binding proteins (32, 34, 39, 44, 45). Thus, we assessed the concentration-dependent effect of MgCl₂ on the enhancement of F_obs with 10 µM TNP-ATP and 5 µM MBP_1.1C (Fig. 3C). MgCl₂ reduced F_obs/F in a concentration-dependent manner to 48% of the control with an EC₅₀ of 61 ± 2 µM, a value virtually identical to K₁ observed with MgATP competition. This result suggests that the MgATP competition curve is composed of both free Mg²⁺ (K₁) and MgATP/ATP anion (K₂) components. Accordingly, the EC₅₀ for MgATP competition of TNP-ATP binding to MBP_1.1C is 3.8 mM (K₂). This EC₅₀ value (K₂) for MgATP competition is consistent with the ~50% reduction in TNP-ATP binding by 5 mM MgATP shown in Fig. 3B and with our previous observations of MgATP inhibition of Kir1.1 channel activity expressed in Xenopus laevis oocytes (EC₅₀ of ~3.5 mM) (24).

We also assessed the effect of a saturating concentration of 1 mM MgCl₂ on TNP-ATP concentration-dependent increases in F_obs with MBP_1.1C (Fig. 3D). Titration of 5 µM TNP-ATP with MBP_1.1C protein in the presence of 1 mM MgCl₂ slightly increased the enhancement factor () to 10.4 ± 0.6 (n = 3; not shown). As shown in Fig. 3D, 1 mM MgCl₂ significantly reduced the F_obs for 0-20 µM TNP-ATP titration of MBP_1.1C. The K_d for TNP-ATP binding to MBP_1.1C increased from 1.9 ± 0.2 (control; F_P;  = 7.7; n = 5) to 12.4 ± 0.6 µM (1 mM MgCl₂; F_{P 1 mm MgCl2};  = 10.4; n = 5; p < 0.01). These results suggest that the Mg²⁺ cation, as well as the MgATP/ATP anion, competes TNP-ATP binding to MBP_1.1C.

We also assessed the ability of NaATP to compete TNP-ATP binding to MBP_1.1C. In contrast to MgATP, NaATP has little effect on the activity of either the Kir1.1 channel expressed in oocytes (24) or the native kidney K_ATP channel (43) at concentrations less than 10 mM. As shown in Fig. 3C, NaATP reduced TNP-ATP fluorescence in a concentration-dependent manner; however, 20 mM NaATP reduced F_obs/F by only 66 ± 2%. The competition data were well fit by either a single-site or a two-site model (Equation 7) yielding an estimated EC₅₀ of 17.5 ± 2.6 mM (Fig. 3C; n = 7). Based on the low affinity of NaATP, the K₂ value for MgATP competition (3.8 mM) was likely due to MgATP complex rather than ATP anion. Thus, the affinity profile for ATP binding to MBP_1.1C is: TNP-ATP (Mg²⁺) MgATP NaATP. The TNP-ATP affinity for some other nucleotide-binding proteins is also higher than for unmodified ATP (34, 39, 46).

TNP-ATP Inhibits the Secretory K_ATP Channel in Principal Cells of Rat Cortical Collecting Duct with Higher Affinity than MgATP-- Given our biochemical evidence for direct binding of TNP-ATP to MBP_1.1C with a higher affinity than MgATP, we assessed TNP-ATP inhibition of native K_ATP channel activity believed to be formed by Kir1.1 (43). Inside-out patches from apical membranes of rat principal cells containing the typical low conductance K⁺ channels (SK) were exposed to varying TNP-ATP concentrations. Fig. 4A shows a representative trace from an inside-out excised apical patch demonstrating that 1 mM TNP-ATP added to the bath (cytosolic side) reversibly inhibited SK channel activity. The TNP-ATP concentration-dependent inhibition of the SK channel is shown in Fig. 4B (n = 6). The EC₅₀ for channel inhibition was 170 µM, a value three to four times lower than for unmodified MgATP (43). This EC₅₀ is consistent with the observed affinity for TNP-ATP binding to MBP_1.1C being greater than for MgATP (Fig. 3C). It is likely, however, that the affinity for TNP-ATP inhibition of the SK channel was underestimated in these experiments because 0.2 mM MgATP (and free Mg²⁺; TNP-ATP competitors) was present in the bath solution to keep these K_ATP channels from running down (43).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   TNP-ATP blocks the small conductance, apical K+ channel in principal cells of rat kidney cortical collecting duct. Collecting ducts were isolated and split open, and the apical membranes patched as described (43). A, Representative single channel current traces from an excised inside-out patch showing that 1 mM TNP-ATP reversibly blocks native KATP channel activity (Vp = 40 mV). c designates the closed channel state. B, TNP-ATP concentration-dependent reduction in fractional K+ channel activity (NPo/NP) in excised membrane patches (n = 6). EC50 was 170 µM. NPo represents the channel activity, in which N is the number of channels in the patch, and Po is the single channel open probability, calculated at a modified filter frequency of 500 Hz by using pCLAMP software (version 6.0.4 of Fetchan and pSTAT; Axon Instruments, Inc.).

The Kinetics and Stoichiometry of TNP-ATP Binding to MBP_1.1 at pH 7.5 and 6.5-- The TNP-ATP to MBP_1.1C protein stoichiometry can be estimated from the TNP-ATP concentration-dependent increases in F_obs shown in Fig. 2B. Using Equation 1 (32), the stoichiometry (N_o) for TNP-ATP binding to MBP_1.1C was 0.89 ± 0.02 mol of TNP-ATP/mol of protein (n = 11). An additional estimate of N_o can be made from the intersection of linear fits to the initial and final F_obs values as suggested by Faller (32). This is possible because F_obs initially increased linearly with 0-1 µM TNP-ATP concentrations, indicating that nearly all of the TNP-ATP was bound to the fusion protein over this range and was flat at TNP-ATP concentration above 15 µM (Fig. 2B, dashed lines; r² = 0.99; n = 11; p < 0.001). The intersection gave a maximal TNP-ATP binding of 4.1 µM at a MBP_1.1C protein concentration of 5 µM (Fig. 2B), yielding a N_o value of 4.1 µM TNP-ATP/5 µM protein or 0.82 mol of TNP-ATP/mol of protein. These N_o values are consistent with a single nucleotide-binding site on MBP_1.1C with 80% of the protein being active.

Assessment of TNP-ATP binding kinetics requires the calculation of bound and free TNP-ATP concentrations and depends on value of  as described by Equation 4. Accordingly, we assessed the  for TNP-ATP binding to MBP_1.1C at pH 7.5 by measuring the protein concentration-dependent increases in F_obs at constant TNP-ATP concentrations of 1 and 5 µM. F_obs increased with increasing MBP_1.1C concentrations in the presence of either 1 or 5 µM TNP-ATP (Fig. 5A). Maximal F_obs (F) values were calculated from exponential fits of the binding data and  calculated according to Equation 3. The  values were similar at 1 and 5 µM TNP-ATP, being 7.4 ± 0.3 (n = 5) and 8.5 ± 0.3 (n = 3), respectively. Thus,  was independent of the fixed TNP-ATP concentration and averaged 7.7 ± 0.3. 

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Enhancement factor (), affinity (Kd), and stoichiometry (N) of TNP-ATP binding to MBP_1.1C at pH 7.5. A, MBP_1.1C protein titration of 1 µm (triangles) and 5 µm (squares) TNP-ATP. Fobs was corrected for protein light scatter. The lines were calculated using an exponential fit, and fluorescence at infinite protein concentration (P) was determined.  was calculated as F/F. The dashed line is the intrinsic fluorescence of 5 µM TNP-ATP in buffer. B, bound TNP-ATP ([B]) plotted against free TNP-ATP ([F]). Bound and free TNP-ATP concentrations were calculated using Equation 4, as described under "Materials and Methods" and Ref. 31. The line was calculated according to Equation 5. C, Scatchard plot for TNP-ATP binding to MBP_1.1C. The line was calculated according to Equation 6.

The  derived from Fig. 5A was used to calculate the bound ([B]) and free ([F]) TNP-ATP concentrations from the data plotted in Fig. 2B as described (31) (Equation 4). A plot of TNP-ATP [B] versus [F] for MBP_1.1C is shown in Fig. 5B and was fit by Equation 5 (r² = 0.98), giving a B_max of 3.8 ± 0.2 µM and a K_d of 2.3 ± 0.2 µM. B_max and K_d were similar to those determined from the F_obs data in Fig. 2B, and the B_max gave a N_o value (3.8 µM TNP-ATP/5 µM protein) of 0.76 mol of TNP-ATP/mol of protein. A Scatchard plot of the TNP-ATP binding data is shown in Fig. 5C, and the data were fit according to Equation 6 (r² = 0.97). The calculated K_d of 2.4 ± 0.1 µM was indistinguishable from that calculated in Figs. 2B and 5B. The calculated stoichiometry (N; Equation 6) was 11.6 ± 0.2 nmol of TNP-ATP bound per mg of protein with a 95% CI of 11.1-12.0. Based on the calculated molecular weight of MBP_1.1C (15.06 nmol/mg), the stoichiometry (mol of TNP-ATP/mol of protein) for TNP-ATP binding to MBP_1.1C was 0.77, ranged from 0.74 to 0.80 (95% CI), and was similar to that derived using Equation 1 from the F_obs data in Fig. 2B.

TNP-ATP binding to MBP_1.1C was significantly enhanced by lowering pH from 7.5 to 6.5 and reducing the salt concentration from 50 mM Tris-Cl to 5 mM MES (Fig. 5). The enhancement factor, calculated from the MBP_1.1C protein titration of 5 µM TNP-ATP, increased from 7.7 ± 0.3 at pH 7.5 (Fig. 5A) to 34.5 ± 1.6 at pH 6.5 (Fig. 6A; n = 5). The K_d calculated from the TNP-ATP concentration dependence of F_obs at pH 6.5 (Fig. 6B; n = 5) using Equation 1 and a  value of 34.5 was 1.0 ± 0.1 µM or less than half of the K_d at pH 7.5. Fit of the Scatchard data by Equation 6 gave a similar TNP-ATP binding affinity of 1.0 ± 0.1 µM (Fig. 6C).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Enhancement factor (), affinity (Kd), and stoichiometry (N) of TNP-ATP binding to MBP_1.1C at pH 6.5. A, MBP_1.1C protein titration of 5 µm TNP-ATP. Fobs was corrected for protein light scatter. The line was calculated using an exponential fit and fluorescence at infinite protein concentration (P) determined.  was calculated as F/F. The dashed line is the intrinsic fluorescence of 5 µM TNP-ATP in buffer. B, TNP-ATP concentration-dependent increases in Fobs with 5 µM MBP_1.1C at pH 6.5. The solid line is the fit according to Equation 1. The intrinsic TNP-ATP fluorescence in buffer at pH 6.5 is shown as FB. The dashed line is fit by a second order polynomial that accounts for the inner filter effect. C, Scatchard plot for TNP-ATP binding to MBP_1.1C. The line was calculated according to Equation 6.

TNP-ATP Binding to the COOH Terminus of Kir6.1-- The binding of TNP-ATP to the COOH terminus, but not the NH₂ terminus, of Kir1.1 (Fig. 2) and the photoaffinity labeling of MBP_1.1C by 8-azido-[-³²P]ATP (Fig. 3A) suggests that the COOH termini of the Kir6.x channels may be sufficient for nucleotide binding (13, 19-23). Thus, we assessed the binding of TNP-ATP to the NH₂ and COOH termini of Kir6.1 [MBP_6.1N and MBP_6.1C, respectively.

Fig. 7 shows that F_obs increased in a TNP-ATP concentration-dependent and saturable fashion with MBP_6.1C (Fig. 7A) but not MBP_6.1N (Fig. 7B) at a pH of 7.5. Both 5 mM MgATP and 4 M urea significantly reduced the increase in F_obs with MBP_6.1C, but urea had little effect on the low F_obs with MBP_6.1N (Fig. 7B). Given that the COOH termini of both Kir1.1 and Kir6.1 bind TNP-ATP, we assessed the specificity for TNP-ATP interactions with K_ATP COOH termini by determining TNP-ATP-dependent increases in F_obs with the COOH terminus of an ATP-insensitive inward rectifier K⁺ channel (Kir2.1; MBP_2.1C) (25). The TNP-ATP concentration-dependent increases in F_obs with MBP_2.1C (Fig 7C) were small and unaffected by 5 mM MgATP or 4 M urea. Thus, unique amino acid sequence(s) specific to the COOH termini of these K_ATP channels determines their ability to bind nucleotides.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   TNP-ATP titration of the MBP fusion proteins of the COOH (C) or NH2 (N) termini of the rat Kir6.1 K+ channel (MBP_6.1C; MBP_6.1N) and the COOH terminus of the ATP-insensitive inward rectifier K+ channel, Kir2.1 (MBP_2.1C) at pH 7.5. TNP-ATP concentration-dependent increases in Fobs in control buffer (squares; FP) and in buffers containing either 5 mM MgATP (inverted triangles; FP MgATP) or 4 M urea (triangles; FP Urea) are shown for (5 µM) MBP_6.1C (A), MBP_6.1N (B), and MBP_2.1C (C). No specific TNP-ATP binding was observed for MBP_6.1N (B) or for MBP_2.1C (D) because FP was low and equal to FP Urea or FP MgATP.

The kinetics of TNP-ATP binding to MBP_6.1C at pH of 7.5 is shown in Fig. 8. MBP_6.1C titration of 5 µM TNP-ATP (Fig. 8A) yielded a  of 17.3 ± 0.7 (n = 3), significantly higher than for MBP_1.1C (7.7 ± 0.3; Fig. 5A). The K_d and stoichiometry for TNP-ATP binding to MBP_6.1C were calculated by fitting the F_obs data (Fig. 8B) to Equation 1 using a  of 17.3 (Fig. 8A): K_d = 3.9 ± 0.4 µM and N_o = 0.82 ± 0.03 mol of TNP-ATP/mol of protein (n = 5). The intercept of linear fits to the initial and final F_obs values (Fig. 8B, dashed lines) gave a similar stoichiometry of 3.6 µM TNP-ATP/5 µM protein = 0.72. The  of 17.3 was used to calculate bound and free TNP-ATP concentrations (Equation 4), and the Scatchard plot of the binding data is shown in Fig. 8C; the K_d calculated from Equation 6 was 3.4 ± 0.3, indistinguishable from that derived using Equation 1 from the F_obs data in Fig. 8B. The TNP-ATP binding stoichiometry derived from Equation 6 was 12.16 ± 0.36 with a 95% CI of 11.3-13.0. Based on the calculated molecular weight of MBP_6.1C (14.33 nmol/mg), the stoichiometry (mol of TNP-ATP/mol of protein) for TNP-ATP binding to MBP_6.1C was 0.87, and ranged from 0.79 to 0.91 (95% CI), and was similar to that derived using Equation 1 from the F_obs data in Fig. 8B.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Enhancement factor (), affinity (Kd), and stoichiometry (N) of TNP-ATP binding to MBP_6.1C at pH 7.5. A, MBP_6.1C protein titration of 5 µM TNP-ATP. Fobs was corrected for protein light scatter. The line was calculated using an exponential fit and fluorescence at infinite protein concentration (P) determined.  was calculated as F/F. The dashed line is the intrinsic fluorescence of 5 µM TNP-ATP in buffer. B, TNP-ATP concentration-dependent increases in Fobs with 5 µM MBP_6.1C at pH 6.5. The solid line is the fit according to Equation 1. The intrinsic TNP-ATP fluorescence in buffer at pH 6.5 is shown as FB. The dashed line is the fit by the second order polynomial that accounts for the inner filter effect. The dotted straight lines were fit to the initial 4 points and final 3 points. The intersection of these lines is indicated (see text for discussion). C, Scatchard plot for TNP-ATP binding to MBP_6.1C. The line was calculated according to Equation 6.

The kinetics of TNP-ATP binding to MBP_6.1C at pH 6.5 is shown in Fig. 9. Similarly to MBP_1.1C (Fig. 6), the fluorescence enhancement factor for TNP-ATP binding to MBP_6.1C at pH of 6.5 was significantly increased over that at pH 7.5:  = 32.8 ± 0.8 (Fig. 9A; n = 5; pH 6.5) versus 17.3 ± 0.7 (Fig. 8A; pH 7.5; p < 0.01). The TNP-ATP concentration-dependent increase in F_obs upon binding to MBP_6.1C at pH 6.5 is shown in Fig. 8B. The F_obs data were well fit by Equation 1 (r² = 0.998) using the  of 32.8 and gave a K_d = 1.0 ± 0.1 µM, a significantly higher affinity than for TNP-ATP binding to MBP_6.1C at a pH of 7.5 (Fig. 8B). The increase in F_obs at pH 6.5 was abolished by 4 M urea (data not shown). The  of 32.8 was used to calculate bound and free TNP-ATP concentrations (Equation 4), and the Scatchard plot of the binding data is shown in Fig. 9C; the K_d calculated from Equation 6 was 1.0 ± 0.1 µM. Thus, TNP-ATP binding to MBP_6.1C exhibits generally similar kinetic characteristics to MBP_1.1C at both pH values of 7.5 and 6.5. 

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Enhancement factor (), affinity (Kd), and stoichiometry (N) of TNP-ATP binding to MBP_6.1C at pH 6.5. A, MBP_6.1C protein titration of 5 µM TNP-ATP. Fobs was corrected for protein light scatter. The lines were calculated using an exponential fit and fluorescence at infinite protein concentration (P) determined.  was calculated as F/F. The dashed line is the intrinsic fluorescence of 5 µM TNP-ATP in buffer. B, TNP-ATP concentration-dependent increases in Fobs with 5 µM MBP_6.1C at pH 6.5. The solid line is the fit according to Equation 1. The intrinsic TNP-ATP in buffer at pH 6.5 is shown as FB. The dashed line is fit by a second order polynomial that accounts for the inner filter effect. C, Scatchard plot for TNP-ATP binding to MBP_6.1C. The line was calculated according to Equation 6.

TNP-ATP Binding to the COOH Terminus of Kir6.C36-- Previous reports (17, 18) have suggested that ATP can directly interact with Kir6.2, based on the photoaffinity labeling of the entire Kir6.2 channel subunit by 8-azido-[-³²P]ATP. In addition, mutations in the COOH terminus alter the EC₅₀ for ATP inhibition of channel activity (13, 19-23). We used the COOH terminus of the functional, ATP-sensitive deletion mutant Kir6.2C36 [MBP_6.2C36] (13) to assess TNP-ATP binding to Kir6.2 (Fig. 10). F_obs values increased in a concentration-dependent manner with MBP_6.2C36 and were significantly enhanced over the buffer (Fig. 10A; F_B) at either pH of 7.5 (Fig. 10A, white squares and dashed line) or 6.5 (Fig. 10A, solid squares and solid line). The TNP-ATP concentration-dependent increases in F_obs were significantly reduced by 5 mM MgATP or 4 M urea (Fig. 10B; pH 6.5 shown; similar results were obtained at pH 7.5 but are not shown). The  value was significantly increased at pH 6.5 to 46.1 ± 0.3 (Fig. 10C; n = 3) compared with 11.4 ± 0.3 at pH 7.5 (Fig. 10C; n = 5). K_d for TNP-ATP binding to MBP_6.2C36 at pH 7.5 and 6.5 calculated using Equation 1 for the F_obs data in Fig. 10A were, at pH 7.5, K_d = 6.8 ± 0.6 µM, N_o = 0.51 ± 0.02 mol of TNP-ATP/mol of protein and, at pH 6.5, K_d = 1.4 ± 0.1 µM. Scatchard plots of the bound and free TNP-ATP concentrations at both pH values are shown in Fig. 10D. K_d and N values calculated using Equation 6 were, at pH 7.5, K_d = 4.9 ± 0.2 µM, n = 6.86 ± 0.14 nmol/mg and, at pH 6.5, K_d = 1.6 ± 0.1 µM. Using the calculated molecular weight of MBP_6.2C36 (15.90 nmol/mg) yielded a stoichiometry (N_o) of 0.43 mol of TNP-ATP/mol of protein (pH 7.5), consistent with one TNP-ATP-binding site/Kir6.2 COOH terminus with ~50% of the protein being active.

View larger version (24K): [in this window] [in a new window]   Fig. 10.   Enhancement factor (), affinity (Kd), and stoichiometry (N) of TNP-ATP binding to MBP_6.2C36 at both pH 7.5 and 6.5. See the legend to Fig. 9 for a general explanation. A, the TNP-ATP concentration-dependent increases in Fobs with 5 µM MBP_6.2C36 (FP) at pH 7.5 (white squares) and pH 6.5 (black squares). The solid lines were calculated according to Equation 1. Fobs was significantly more enhanced at pH 6.5 than at 7.5. Intersection of linear fits of initial and final TNP-ATP concentrations (dotted lines) is shown (see text for discussion). For comparison, Fobs is shown for TNP-ATP in buffer (diamonds and dashed line, second order polynomial). B, denaturing the MBP_6.2C36 fusion protein at pH 6.5 with 4 M urea (black inverted triangles and dashed line; FP Urea) or addition of 5 mM MgATP (black triangles and dashed line; FP MgATP) significantly reduced the increases in Fobs. C, MBP_6.2C36 protein titration of 5 µM TNP-ATP at pH values of 7.5 (diamonds) and 6.5 (squares). Fobs was corrected for protein light scatter. The lines were calculated using an exponential fits, and fluorescence at infinite protein concentration (P) was determined.  was calculated as F/F. The gray bar represents the intrinsic fluorescence of 5 µM TNP-ATP in buffers at pH of 7.5 and 6.5. D, Scatchard plots for TNP-ATP binding to MBP_6.2C36 at pH of 7.5 (squares) and 6.5 (diamonds). The lines were calculated according to Equation 6.

Multimerization Potential of MBP_1.1C, MBP_6.1C, and MBP_6.2C36-- K_ATP channel pores are formed of four identical Kir subunits (47, 48). To assess whether the COOH termini of MBP_1.1C, MBP_6.1C, and MBP_6.2C36 proteins have the capacity to self-assemble into oligomers in the absence of the NH₂ termini and transmembrane spanning segments and the pore, we analyzed dilute solutions of these fusion proteins by SDS-PAGE in the presence of dithiothreitol (DTT) followed by Western blotting using anti-MBP as described (42). In the absence of cross-linking agents and disulfide bond formation, the three fusion proteins exhibited oligomeric structures (Fig. 11, A, C, and D, first lanes). Oligomerization was enhanced with cross-linking using glutaraldehyde (Fig. 11). At concentrations of glutaraldehyde of 0.005-0.025%, the trimer and tetrameric forms became dominant. At high concentrations of glutaraldehyde (0.05%), higher order multimers were produced that either did not enter the gel or migrated near the top of the gel. The oligomerization of these proteins was specific for the COOH termini because MBP has been shown not to oligomerize with glutaraldehyde concentrations up to 1% under our conditions (42).

View larger version (71K): [in this window] [in a new window]   Fig. 11.   Multimerization of MBP fusion proteins containing the COOH termini of Kir1.1 (MBP_1.1C), Kir.6.1 (MBP_6.1C), and Kir6.2C36 (MBP_6.2C36). A, C, and D show the effects of glutaraldehyde cross-linking on multimer formation. 0.15 µg of the purified MBP fusion proteins were incubated with different concentrations of glutaraldehyde as indicated and then analyzed by Western blotting using anti-MBP antibody. The proteins were resolved by 7.5% SDS-PAGE. Molecular mass of makers are shown (in kDa). M, monomer; D, dimer; R, trimer; T, tetramer. B, reducing agents, DTT (1 mM) + -ME (10 mM), do not affect TNP-ATP binding to MBP_1.1C at pH 7.5. TNP-ATP concentration-dependent increases in Fobs in the absence (DTT/-ME) and presence (+DTT/-ME) of reducing agents.

Although oligomerization of the COOH termini of these fusion proteins does not depend on disulfide bridge formation, Kir1.1 channels are redox-sensitive with pH-mediated channel closure resulting in exposure of a COOH-terminal cysteine (Cys³⁰⁸) that forms a disulfide bond and locks the channel in the closed state (49). Therefore, we examined whether reducing agents alter TNP-ATP concentration-dependent increase in F_obs with MBP_1.1C. One mM DTT with 10 mM -ME did not significantly change the K_d for TNP-ATP binding to MBP_1.1C (Fig. 11B): DTT/-ME, 2.7 ± 0.3 µM, n = 23; +DTT/-ME, 1.8 ± 0.2 µM, n = 3.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our results provide direct evidence for high affinity TNP-ATP binding to the cytosolic COOH-terminal domains of the pore-forming subunits of K_ATP channels: Kir1.1, Kir6.1, and Kir6.2C36. NH₂ termini of the Kir1.1 and Kir6.1 K_ATP channels did not bind TNP-ATP, demonstrating that the nucleotide-binding domain is restricted to COOH termini. A summary of TNP-ATP binding to these COOH termini is shown in Fig. 12. Fig. 12A shows the relative increases in F_obs/F for all three COOH termini at both pH 7.5 and 6.5. The higher affinities for TNP-ATP binding at pH 6.5 are apparent. Fig. 12B shows the Scatchard plots and summaries of the stoichiometry (N_o) and K_d values. The TNP-ATP affinity profile at pH 7.5 was: MBP_1.1C > MBP6.1C > MBP6.2C36. At a pH of 6.5, however, the K_d values for all three proteins were similar at ~1 µM. Reducing pH to 6.5 also increased the enhancement factor () for TNP-ATP binding.

View larger version (26K): [in this window] [in a new window]   Fig. 12.   Summary of TNP-ATP binding to MBP_1.1C, MBP_6.1C, and MBP_6.2C36 at pH values of 7.5 and 6.5. A, fractional (F/Fmax) increases in Fobs with increasing concentrations of TNP-ATP at pH 7.5 (black symbols and solid lines) or pH 6.5 (white symbols and dashed lines). B, Scatchard plots of the binding data for all proteins at both pH values. Stoichiometries (No; mol of TNP-ATP b