Scholarly article on topic 'Intramolecular Interaction of SUR2 Subtypes for Intracellular ADP-Induced Differential Control of KATP Channels'

Intramolecular Interaction of SUR2 Subtypes for Intracellular ADP-Induced Differential Control of KATP Channels Academic research paper on "Biological sciences"

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Academic research paper on topic "Intramolecular Interaction of SUR2 Subtypes for Intracellular ADP-Induced Differential Control of KATP Channels"

Intramolecular Interaction of SUR2 Subtypes for Intracellular ADP-Induced Differential Control of Katp Channels

Kenji Matsushita, Kengo Kinoshita, Tetsuro Matsuoka, Akikazu Fujita, Takashi Fujikado, Yasuo Tano, Haruki Nakamura, Yoshihisa Kurachi

Abstract—ATP-sensitive K+ (KATT) channels are composed of sulfonylurea receptors (SURs) and inwardly rectifying Kir6.2-channels. The C-terminal 42 amino acid residues (C42) of SURs are responsible for ADP-induced differential activation of Katp channels in SUR-subtypes. By examining ADP-effect on Katp channels containing various chimeras of SUR2A and SUR2B, we identified a segment of 7 residues at central portion of C42 critical for this phenomenon. A 3-D structure model of the region containing the second nucleotide-binding domain (NBD2) of SUR and C42 was developed based on the structure of HisP, a nucleotide-binding protein forming the bacterial Histidine transporter complex. In the model, the polar and charged residues in the critical segment located within a distance that allows their electrostatic interaction with Arg1344 at the Walker-A loop of NBD2. Therefore, the interaction might be involved in the control of ADP-induced differential activation of SUR2-subtype Katp channels. (Circ Res. 2002;90:554-561.)

Key Words: homology modeling___ATr

■ sulfonylurea receptor

Katp channel

nucleotide binding domain Walker motif

ATP-sensitive K+ (Katp) channels are inhibited by intracellular ATP and activated by nucleoside diphosphates and thus provide a link between the metabolic state an cellular excitability in various organs, including pancreas, heart, vasculature, and brain.1 These channels are associated with such cellular functions as insulin secretion, cardiac preconditioning, vasodilatation, and neurotransmitter release KATP channels are heterooctamers composed of an AT binding cassette protein, known as the sulfonylurea rec (SUR), and an inwardly rectifying K+ channel (Kir) sub Kir6.0.2,3 Three kinds of SUR, SUR1, SUR2A, and SUR2B, have been isolated and their roles in formation of functional K+ channels with Kir6.0 subunits have been extensively examined.2-6 It is now widely accepted that SUR1 and SUR2B represent pancreatic, cardiac, and vascular smooth muscle types of SUR, respectively. When expressed with Kir6.2, all 3 subtypes of SUR form Katp channels that exhibit the same single-channel characteristics; weak inward-rectification and a unitary conductance of ^80 pS in the inward-direction with 150 mmol/L extracellular K+. They, however, show distinct sensitivities to different vasorelaxant K+ channel opener compounds (KCOs), intracellular MgADP, and sulfonylurea drug derivatives.7-11 The different pharmacological sensitivities depend on the SUR-subtype. SUR has been assigned to the ATP-binding-cassette (ABC)

superfamily, as is assumed to possess 17 transmembrane segments and 2 nucleotide-binding domains (NBDs) with

: have no

hand re

Walker-A and -B consensus motifs for binding intracellular nucleotides.12,13 However, structural elements of SURs and their functional roles responsible for their different features have not been fully determined.

majority of ABC proteins are active transporters, ilizing the energy of ATP hydrolysis to pump solutes and substances across the membrane. SUR on the other regulates the behavior of Kir6.0 channel pores, and thus, the role of the 2 NBDs differs from those of other ABC-proteins.12 In SUR, it is thought that NBD1 binds MgATP and that NBD2 binds MgADP and/or binds and hydrolyzes SUR2A, MgATP. The binding of MgADP to NBD2 causes activation of Katp channels.9,14-15 Depending on the subtype of SUR, the sensitivity of KATP channels to MgADP is divergent. KATP channels containing SUR1 or SUR2B can be effectively activated by ADP, whereas those containing SUR2A require much higher concentrations of ADP. SUR2A and SUR2B are generated from a single gene and differ only in their splicing site, which is the 42 amino acid residue C-terminal tail (C42).5 The C42 region of SUR2B shares ~30% amino acid sequence homology with that of SUR2A but ^70% with that of SUR1. Therefore, C42 should play a critical role in the SUR subtype-dependent activation of KATP channels by

ll ili

Original received October 23, 2001; revision received January 29, 2002; accepted January 30, 2002.

From the Departments of Pharmacology II (K.M., T.M., A.F., Y.K.) and Ophthalmology (K.M., T.F., Y.T.), Graduate School of Medicine, and the Research Center For Structural Biology (H.N.), Institute for Protein Research, Osaka University, Suita, Osaka, Japan; and the Division of Science of Biological Supramolecular Systems (K.K.), Graduate School of Integrated Science, Yokohama University, Yokohama, Kanagawa, Japan.

Correspondence to Y. Kurachi, Dept of Pharmacology II, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail ykurachi@pharma2.med.osaka-u.ac.jp © 2002 American Heart Association, Inc.

Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000012666.42782.30

MgADP.16 Recently, we showed that C42 of SUR2A, but not that of either SUR2B or SUR1, was able to reduce stimulation of Katp channels by ADP acting via NBD2.17

The purpose of this study is to obtain further insight into the role of C42 on Katp channel-activation by ADP via NBD2, by using electrophysiological and 3-D structural model analyses with different chimeras of SUR2A and SUR2B. We found that the middle segment of C42 composed of 7 amino acids are critical for differential effects of ADP on SUR2-subtype Katp channels. In the 3-D models, the polar and charged residues in the segment located within a distance that allows their electrostatic interaction with Arg1344 on the Walker-A loop of NBD2.

Materials and Methods Molecular Biology

The cDNAs of mouse Kir6.2 and SURs were used.5,11 The coding region of each cDNA was individually subcloned into an expression vector, pcDNA3 (Invitrogen, San Diego, Calif). All chimeric cDNAs and cassette mutants were formed between C42s of SUR2A and SUR2B. Chimeric cDNA and mutant constructs were produced using the Splicing by Overlap Extension (SOE) PCR technique as previously described29 and Quick Change Site-Directed Mutagenesis Kit (STRATAGENE). All chimeric and mutated SURs were confirmed by DNA sequencing.

Functional Coexpression of SURs an Kir6.2 cDNAs

The plasmid containing Kir6.2 was cotransfected with wild-type or chimeric SURs into human embryonic kidney (HEK) 293T cells using LipofectAMINE (Life Technology, Inc). To monitor the efficiency of transfection, pCA-GFP (S65A) was also cotransfected. The cells expressing green fluorescent protein (GFP) were identified by fluorescence microscopy and used for electrophysiology

Journal of the American Heart Association

Electrophysiology

The channels expressed in the cotransfected HEK293T cells were recorded in the inside-out configuration of the patch clamp tec nique. The tip of pipettes were coated with Sylgard and heat-polished. The bath was perfused with a solution containing (in mmol/L) 150 KCl, 5 EGTA, 2 MgCl2, and 5 HEPES-KOH (pH

7.3), in which the concentration of free Mg2+ was adjusted to [ 1.4 mmol/L. Pipettes were filled with a solution containing (in mmol/L) 150 KCl, 1 MgCl2, 1 CaCl2, and 5 HEPES-KOH (pH

7.4). Single channel ion currents were recorded in excised membrane patches voltage-clamped at —60 mV membrane potential. All experiments were performed at room temperature (^25°C). The data were recorded on videocassette tapes with a PCM converter system (VR-10B, Instrutech Corp). They were reproduced, low-pass filtered at 1 kHz (—3 dB) by an 8-pole Bessel filter (Frequency Devices), sampled at 5 kHz, and analyzed off-line on a computer (Macintosh G3, Apple Computer Inc) using commercially available software. The channel activity was expressed as relative NPo (rNPo) with reference to the maximum NP0 measured in the absence of intracel-lular nucleotides in each inside-out patch. All data were derived from at least 6 distinct patches and expressed as mean±standard error (SE).

Homology Modeling and Validation of Second Nucleotide Binding Domains in SUR2s

The whole cytosolic region containing second nucleotide-binding domain (NBD2) and C42 of SUR2A and SUR2B were determined by knowledge-based homology modeling using the program Mod-eler4.19 The input consisted of a sequence alignment of the SUR-NBD2/C42s and HisP or MalK and MJ1267, and coordinates obtained from the Rutgers Protein Data Bank for crystal structure of

HisP, MalK, and MJ1267 (PDB ID: 1B0U, 1G29, and 1G6H, respectively). ADP was modeled by coordinating it to the equivalent residues that were determined for HisP. Models in figures were prepared using InsightII2000 (Molecular Simulations Inc). Quality of models were determined by stereochemical properties (using PROCHECK20) and root mean standard deviation calculated in InsightII2000.

Results

The Responses to Intracellular ADP of ATP-Sensitive K+ Channels Containing Different SUR2 Subtypes and Homology Models of Their C-Terminal Cytosolic Regions

The responses of ATP-sensitive K+ (Katp) channels composed of Kir6.2 and either SUR2A or SUR2B are shown in Figure 1A. HEK293T cells were cotransfected with Kir6.2 and one of the wild-type SUR2s. On formation of inside-out patches vigorous Katp channel activity appeared in both cases. ATP (1 mmol/L) added to the internal solution almost completely suppressed channel activity. The activity of SUR2A/Kir6.2 channels was only weakly enhanced by ADP;

dADP exhibited practically no effect at 30 ^mol/L and enhanced channel activity only weakly at 300 ^mol/L. Even with 1 mmol/L ADP, the relative NPo of SUR2A/Kir6.2 channels was ^0.13 of the maximum activity. On the other hand, ADP effectively enhanced SUR2B/Kir6.2 channel-

endent fashion (see also the top luate the responses to ADP of Katp channels containing the differential type of SUR, we defined 2 parameters, Imax and EC50. Imax is the maximum value of Katp channel activity induced by ADP with reference to that in the absence ofnucleotides; EC50 is the concentration f ADP at the half-maximum channel activity. The Imax value the wild-type SUR2A/Kir6.2 channel was ^0.15, which around one third of that (~0.43) of wild-type SUR2B/ Kir6.2. The EC50 value for SUR2A/Kir6.2 was 250 ^mol/L DP, which was ^9 times larger than that for SUR2B/Kir6.2 (Table).

Most ABC proteins bind intracellular nucleotides by using recognition of phosphates at the Walker-A segment of a nucleotide-binding domain. The C42-region is located ^150 amino acids away from Walker-A, and thus, a specific interaction between the 2 segments was not obvious (Figure 1B). Therefore, we developed 3-D structure models of the cytosolic C-termini of SUR2A and B that contain both NBD2 and C42 with the homology modeling technique. We adopted the structure of HisP as the template because of the following reasons. (1) HisP is an intracellular component of Histidine permease, an ABC superfamily protein whose 3-D crystal structure is available at the atomic level.18 (2) Histidine permease binds nucleotides at its Walker-motifs that correspond to those in SURs. (3) Alignment between HisP and SUR2-NBD2/C42 shows high conservation of some motifs and overall identity of 21% in the region. Furthermore, the secondary structures of the region containing NBD2 and C42 in SUR2s are predicted to show high similarity to that of HisP (Figure 1B). Therefore, it seemed reasonable to construct homology models of the entire C-terminal cytosolic regions of SUR2s based on the HisP structure. These models were developed using MODELLER4 (Figure 1C).19 The geomet-

might be conserved among various ABC transporters, and the developed models based on HisP may be usable for present analyses. In the models of both SUR2A and SUR2B, C42 was located close to the Walker-A segment of NBD2.

Structure-Based Chimeric Study of the Effect of the C42 Region on Katp Channel Activity

To identify the critical part of C42, we constructed chimeric SURs of SUR2A and SUR2B by dividing their C42 into 3 portions (Figure 2A). Because the homology models predicted that 2 putative ^ strands (^11 and p12 with a turn) in C42 were located very close to the Walker-A in NBD2, the middle portion used to construct chimera was designed to contain both fi11 and ^12. The designation AAA indicates the 3 sections of C42 to consist of wild-type SUR2A, whereas BBB represents those of wild-type SUR2B. The left portion (A.. or B..) was composed of amino acids 1505 to 1510, the middle portion (.A. or .B.) was amino acids 1511 to 1529, and

1 ■ -a Fighting Heart Disease i ;l Stroke . • 1 1 r r\ -t r a s

the right portion (..A or ..B) amino acids 1530 to 1546. Chimeras constructed on this plan are shown in Figure 2A.

Figure 2B shows examples of the responses of KATP hannels composed of 2 chimeric SURs (ABA and BAB) and Kir6.2 to ADP. Figure 2C summarizes the results obtained from the different chimeric SUR2s. When the middle portion of C42 was adopted from SUR2A (.A.), the KATP channel activity induced by ADP was almost similar to that of

Figure 1. Differential effects of intracellular ADP on Katp channels composed of splice variants of SUR and Kir6.2 and homology models of the cytosolic C-termini of SUR2. A, Traces of Katp channel currents recorded from excised inside-out membrane patches of HEK293T cells transfected with Kir6.2 and either wild-type SUR2A (left) or wild-type SUR2B (right). I/O ir cates when patches were excised from the cells. The bars above the traces indicate periods for which the intracellular fac of the patches was superfused with solution that contained ATP and aDp. B, Sequence alignment of the cytosolic C-termini of SUR2A and SUR2B (amino acids 1305 to 1546) and the corresponding region of HisP. Predicted secondary structure of the alignment indicated beneath, (a-helices are colored light green and /3-strands colored blue.) Some conserved motifs (Walker-A, Walker-B, Q-loop, D-loop, signature, and switch) are boxed in —

orange. The C42 region is enclosed in the red box. C, Models were built based on the X-ray crystal structure of HisP18 using Modeler 419 and show the Walker-A motif in blue and the C42 regions of SUR2A and SUR2B in magenta and light green inside of the square and orange outside of the square.

rical qualities of the models were examined by a program PROCHECK, which provided the acceptable high scores for the developed models (see online Figure 1B that can be found in the online data supplement available at http://www.circresaha. org and the following results of PROCHECK).20 The SUR-NBD2/C42 models were also constructed using the structures of other nucleotide-binding proteins, MalK and MJ1267, as the templates for evaluation of the models constructed on HisP.21,22 The details of this examination are also provided in the online data supplement. These evaluations indicated that the SUR2-NBD2/C42 models developed from HisP, MalK, and MJ1267 structures were very similar from each other. Thus, the structure of the region containing NBD2 and C42

Figure 2. Influence of the C42 region on activation of Katp channels by ADP. A, Schematic diagram of wild-type and chimeric constructs of the C42 region of SUR2. Top line indicates the predicted secondary structure based on the HisP model. AAA indicates wild-type SUR2A; BBB, wild-type SUR2B. The 3 letters represent amino acids 1505 to 1510, amino acids 1511 to 1529, and amino acids 1530 to 1546, respectively. B, Traces of Katp channel currents recorded from excised inside-out membrane patches. Currents were recorded from Katp channels composed of chimeric SUR molecules ABA (left) and BAB (right). Patches were superfused with solutions that contained ATP and ADP as indicated by the bars above the traces. C, Relationship between the concentration of ADP and relative NPo (rNPo) of Katp channels containing chimeric SUR. The 3-letter code corresponds to the chimeric constructions of the C42 region of SUR2 illustrated in (A). Relative NPo is expressed as mean±SE of results obtained from at least 6 isolated patches.

Parameters for the Evaluation of the Response to Intracellular ADP of KAtp Channel

SUR (max EC50

2A 0.15 250

2B 0.43 28

AAB 0.20 140

BAA 0.18 200

BAB 0.21 130

ABB 0.37 26

BBA 0.37 17

ABA 0.39 50

AaabA 0.18 330

AbaaA 0.15 300

AbabA 0.16 170

BbabB 0.20 140

BbbaB 0.30 17

BabbB 0.31 33

AabaA 0.42 45

BabaB 0.32 4258

(.b.), the KATp channels were effectively activated by as little as 30 ^mol/L ADP (BabaB and AabaA in Figure 3B and the bottom panels in Figure 3C; Table) and thus corresponded to the behavior of the entire wild-type SUR2B. It is worthy of note that chimera AabaA, which consisted of SUR2A except for amino acids 1516 to 1525, showed a response to ADP typical of SUR2B, although the equivalent chimera of SUR2B, BbabB, showed a reaction to ADP typical of SUR2A. It is thus clear that the 10 amino acid residues 1516 to 1525, which include the turn between p11and p12, are essential for differential activation of KATP channels by ADP.

Critical Residues in the 1516 to 1525 Amino Acid Region of SUR

When the 10 amino acid residues in positions 1516 to 1525 are compared between SUR2A and SUR2B, there are 2 characteristic polar and charged residues at positions 1517 and 1518 in p 11. In SUR2A, they are polar Ser (S) and

Fighting Heart Disease and Stroke

negatively charged Glu (E), and positively charged Lys (K)

2A(SE1517KR)

2B(KR1517SE)

2B(R1518A)

2B(K1517A)

Aab'a'a'aA

Aab'b'a'aA

Aab'a'b'aA

Aab'b'a'aA(M1516F)

e 50 ar<

0.27 97

0.15 210

0.29 0.13 0.15

13 170 190

Boldface type indicates SUR2A type; regular, SUR2B type. /max indicates the maximum response of Katp channel activity induced by Al (rNPo); EC50, the ADP concentration at the half-maximal activation of ch; (^mol/L).

Table shows the name of wild-type and chimeric SURs and their parameters, /max and EC50. Each parameter is defined (see the text in detail), and values wei calculated by curve fitting using Hill equation (rNPo=[/max/{1+EC5l/[ADP])n}]). The Hill coefficient (n) for all curves was (between 0.7 and

ire }]).

TCippinc

wild-type SUR2A/Kir6.2 channels (AAA) in 7max and^O™. On the other hand, when the portion was from SUR2B(VB.), the channel's response was similar to that of KAIP channelsf containing wild-type SUR2B (Table). The middle portion of C42 thus appeared to be mainly responsible for the different activation of KATP channels by ADP.

The middle portion of C42 (amino acids 1511 to 1529) was further dissected. It was divided into 3 parts with each part being designated as "a" if derived from SUR2A and designated as "b" if derived from SUR2B (Figure 3A). The first part (a.. or b.. ) consisted of amino acids 1511 to 1515, the second part (.a. or .b.) consisted of amino acids 1516 to 1525, and the third part (..a or ..b) consisted of amino acids 1526 to 1529. KATP channels containing chimeric SURs where the second part of middle portion of C42 consisted of 2A type (.a.) were only slightly activated by high concentrations of ADP (AbabA and BbabB in Figure 3B and the top panels in Figure 2C) and thus corresponded to the behavior of the entire wild-type SUR2A (Table). On the other hand, when the second part of middle portion of C42 consisted of 2B type

Figure 3. Influence of the central part of the C42 region on the activation of Katp channels by ADP. A, Schematic representation of the C42 region of SUR2 where the central portion (amino acids 1511 to 1529) was divided into 3 parts corresponding to amino acids 1511 to 1515, 1516 to 1525, and 1526 to 1529. The letter code "a" and "b" corresponds to constructs obtained from SUR2A and SUR2B, respectively. B, Traces of Katp channel currents and their reactions to intracellular ATP and ADP obtained from 4 chimeric constructs. Bars and labels are as for Figure 1. C, Relationship between the concentration of ADP and relative NPo (rNPo) of Katp channels containing chimeric SUR. The letter code identifies chimeric constructs of the C42 region shown in (A).

charged residues, especially Arg 1518, in ^11 of SUR2B are necessary but not sufficient to impose the high efficacy and sensitivity of KAtp channels to MgADP.

The next section therefore attempted to determine what other amino acids in this region of SUR2B needed to be added to the positively charged Lys and Arg to impose high efficacy and sensitivity to ADP to SUR2A. The 10 amino acid residues in positions 1516 to 1525 were divided into 3 segments. Segment 1 consisted of amino acids 1516 to 1518, segment 2 consisted of amino acids 1519 to 1522, and segment 3 consisted of amino acids 1523 to 1525. A schematic representation of the constructed mutants is shown in Figure 5A where segments are designated as a' or b' representing residues derived from SUR2A or SUR2B, respectively. The addition of Met at position 1516 to Lys and

Arg at positions 1517 and 1518 (chimera Aab'a'a'aA) was American Heart tfA Association®^^

Figure 4. Influence of charged residues in the central part of the C42 region on the activation of Katp channels by ADP. A, Schematic diagram illustrating point mutations of polar and charged residues in the central part of the SUR2 C42 region. Amino acids 1517 and 1518 are polar and negatively charged in SUR2A and positively charged in SUR2B. B, Reaction of tl indicated mutated Katp channels to intracellular ATP and Al are shown as single channel currents (left) and graphs of relati NPo and ADP concentration (right). C, Sites of possible interaction between the C42 region and the Walker-A loop in the mod els of SUR2. Two polar and charged residues (red in SUR2A and blue in SUR2B) are closely located beneath R1344 (blue ball and stick) of the Walker-A segment. The hydrophobic residue (green ball and stick) before the 2 charged residues seeiti^ to touch the Walker-A segment.

and Arg (R) in SUR2B. From the 3-D model, these residues are expected to be closely located to a positively charged residue Arg (R) on the Walker-A motif in NBD2 (Figure 4C) because the distance in the model between each Cfi ranged from 7.3- to 8.9-A. Therefore, we constructed mutant SURs where these charged residues were either exchanged between SUR2A and SUR2B or altered to Ala (Figure 4A). Exchanging positively charged KR for polar and negatively charged SE in SUR2B resulted in behavior similar to SUR2A (Figure 4B and Table). But, exchanging SE for KR in SUR2A had no effect, and the behavior of the KAtp channels remained typical of SUR2A. Canceling the charge at position 1518 in SUR2B abolished the high efficacy and sensitivity to ADP of the channels while canceling that at position 1517 showed less effect (Table). These results suggest that the positively

Figure 5. Identification of partner residues necessary for the effect of polar and charged residues on ADP-mediated activation of Katp channels. A, Schematic diagram representing further chimeric dissection of the central part of the C42 region of SUR2. The central section (amino acids 1516 to 1525) was divided into 3 segments, amino acids 1516 to 1518, 1519 to 1522, and 1523 to 1525. The designations a' and b' represent constructions corresponding to SUR2A and SUR2B, respectively. B, Reactions of the indicated mutated Katp channels to ATP and ADP are shown as single channel currents (top) and graphs of relative NPo and ADP concentration (bottom). C, Illustrations of the C42 region and the Walker-A segment in models. The 2 charged residues in the C42 region are closely located beneath R1344 of the Walker-A segment. The surrounding hydrophobic residues seem to connect the Walker-A segment to the outside surface of NBD2. Table of partner residues is shown in right panel.

not sufficient to evoke high efficacy and sensitivity to ADP in SUR2A. Further addition of segment 3 from SUR2B (chimera Aab'a'b'aA) was also without effect. However, a chimera that contained segments 1 and 2 from SUR2B (chimera Aab'b'a'aA) was able to reconstitute the response of SUR2B to ADP in the SUR2A backbone (Figure 5B and Table). This effect was abolished when the first residue of these 2 segments, Met1516, was replaced by the equivalent Phe from SUR2A (chimera Aab'b'a'aA [M1516F]). Thus, the 7 residues 1516 to 1522 in C42 appeared to be necessary and enough to confer 2B-type response to SUR2A.

Charged Residues in the Walker-A Motif of NBD2 Also Play a Critical Role in ADP-Mediated Channel Activation

The next question we addressed was how the positively charged residues, Lys and Arg, in C42 of SUR2B control the function of the Walker-A loop in NBD2. There exist 2 positively-charged residues in the Walker-A, Arg1344 and Lys1348 (Figure 6A). Lys1348 is thought to be the binding site of ADP to the Walker-A,9,14,15,23-26 whereas Arg1344 is located near the N-terminal edge of the Walker-A and is not thought to be directly involved in ADP binding. The homol-ogy model indicated that this Arg residue located within the distance to interact with the positively charged residues, Lys and Arg, in SUR2B-C42 (Figure 5C and Figure 6A). The mutation of Arg1344 to Ala caused as large reduction of ADP-induced channel activity as that of mutating Lys1348 to Met (Figures 6B and 6C). Therefore, Arg1344 may be somehow functionally involved in control of Walker-A func tion such as ADP binding.

Discussion

This study has revealed that a segment of 7 amino acids in th middle portion of C42 is critical for the SUR2 subtype-dependent, ADP-induced differential activation of KAIP channels. The homology structure models developed based on HisP structure suggest that the polar and charged residues in the segment locate within a distance that allows electrostatic interaction with Arg1344 at Walker-A of NBD2. In this study, channel activity was used as a functional readout to arrive at a mechanistic explanation for SUR function. Thus, we could not identify the function of NBD2 responsible for the ADP-induced alteration in channel activity. The candidates may include ADP binding at Walker-A, ATPase-activity at NBD2, and control of interaction between SUR and Kir6.2.26 Because it was reported that NBD2 of SUR2B exhibits higher binding capability to ADP than that of SUR2A27 and because the primary function of Walker-A loop is to bind nucleotides, it seems likely that the difference between SUR2A and SUR2B in the binding property of ADP to their NBD2 may be one of the most possible candidates. However, further studies are needed to identify the function of NBD2 responsible for the ADP-induced alteration of KATP channel activity.

The polar and charged residues in the middle portion of C42 may be critical to control the sensitivity of NBD2 to

Figure 6. Influence of basic residues in the Walker-A segment on the activation of Katp channels by ADP. A, Mutations of charged residues in the Walker-A loop of NBD2 of SUR2B are indicated schematically (left) and indicated in the model (right). B, Single-channel currents from Katp channels bearing the indi-

ed mutations to SUR2B during superfusion with ATP and DP as indicted by the bars above the traces. C, Effect of ADP on relative NPo (rNPo).

ADP. Because in the homology model, the polar and charged residues located within a distance that allows electrostatic interaction with Arg1344 at Walker A, one possibility is that the conformation of the Walker-A loop might be affected by the interaction. In SUR2A, the residues are polar Ser and negatively charged Glu, whereas they are positively charged Lys and Arg in SUR2B. Therefore, the possible electrostatic interaction between Arg1344 and C42 is expected to be in the opposite sense between SUR2A and SUR2B, which might be important for the SUR2 subtype-dependent differential effects of ADP on KATP channels. It was found, however, that the polar and charged resides in C42 were not sufficient to explain the difference (Figure 4). A segment of 7 amino acid residues of SUR2B containing the polar and charged residues were found necessary to increase the sensitivity of SUR2A to ADP (Figure 5C). The homology models showed that this segment would form a ^-turn. The head of the segment (Phe in SUR2A, Met in SUR2B) was located very close to the back of the Walker-A segment in the model (Figures 4C and 5C). Thus, the ^-turn and Walker-A would interact sterically over a short distance. A number of amino acids surrounding the polar and charged residues in the segment are hydrophobic

and thus might also have a steric effect for arranging the positions of the polar and charged residues to locate within appropriate distance from Walker-A.

The candidate amino acid at Walker-A loop for possible electrostatic interaction with the polar and charged residues in C42 was identified Arg1344 in the model. Although Arg1344 localized at the edge of Walker-A is thought not to be directly involved in the binding of ADP to the loop, SUR2B(R1344A) abolished ADP-induced KAIP channel activation as the mutant at the putative ADP-binding site in Walker-A, SUR2B(K1348M). This result may be in line with the notion that the polar and charged residues in C42 were within the distance that allows electrostatic interaction with Arg1344. Therefore, one possibility is that the electrostatic interaction between the polar and charged residues in C42 and Arg1344 is involved in the control of SUR2 subtype-dependent, ADP-induced differential activation of KAtp channels. Resolution of the actual protein structure of SUR, however, is needed to further examine this possibility.

The roles of some of the structural elements in both SUR and Kir6.0 in the behavior of functional KAIP channels have been clarified. The transmembrane domains of SUR1 are needed for the interaction with Kir6.2, whereas the first transmembrane segment (M1) and the N-terminus of Kir6.2 are involved in the interaction with SUR.28 N-terminus of Kir6.2 is also critically involved in the control of ch; gating,29 because both cytosolic N- and C-termini of Kir6 interact to form the ATP-binding pocket for closure of KAIP channels.30-34 In native KAIP channel proteins C-terminus of SUR1 and N-terminus of Kir6.2 are closely located.34 Therefore, alteration in the conformation of C-terminus of a SUR induced by ADP action on NBD2 at Walker-A may affect channel gating via its interaction with Kir6.2 N-terminus. 1123.. Recently, a dramatic alteration in the conformation of NBD especially at the linker region between Walker-A and -B motifs (signature sequence) by nucleotide binding to Walker-A was predicted in the crystallization analysis of ADP-bound MJ1267.22 Thus, alteration in the conformation 15 of NBD2 induced by ADP binding to Walker-A might be an indispensable step in the ADP-induced activation of KAIP channels. Further studies are needed to clarify in SUR2-subtypes the relation between the ADP-induced alteration in the conformation of NBD2 and the critical segment in the center portion of C42.

Acknowledgments

This work was supported by the Grant-in-Aid for Specific Research on Priority Area (B) (12144207) (to Y.K.) from the Ministry of Education, Science, Sports and Culture of Japan, by a grant from the Research for the Future Program of the Japanese Society for the Promotion of Science (96L00302) (to Y.K.), by "Research Grant for Cardiovascular Disease" (11C-1) from the Ministry of Health and Welfare of Japan (to Y.K.), and by a grant from Vehicle Racing Commemorative Foundation (to Y.K.). We thank Dr Ian Findlay for his critical comments on this article.

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Inagaki N, Gonoi T, Clement JP IV, Namba N, Inazawa J, Gonzalez G, Aguilar-Bryan L, Seino S, Bryan J. Reconstitution of IKatp: an inward rectifier subunit plus the sulfonylurea receptor. Science. 1995;270: 1166 -1170.

Inagaki N, Gonoi T, Clement JP IV, Wang C-Z, Aguilar-Bryan L, Bryan J, Seino S. A family of sulfonylurea receptors determines the pharmacological properties of ATP sensitive K+ channels. Neuron. 1996;16: 1011-1017.

Isomoto S, Kondo C, Yamada M, Matsumoto S, Higashiguchi O, Horio Y, Matsuzawa Y, Kurachi Y. A novel sulfonylurea receptor forms with BIR (Kir6.2) a smooth muscle type ATP-sensitive K+ channel. J Biol Chem. 1996;271:24321-24324.

Yamada M, Isomoto S, Matsumoto S, Kondo C, Shindo T, Horio Y, Kurachi Y. Sulphonylurea receptor 2B and Kir6.1 form a sulphonylurea-sensitive but ATP-insensitive K+ channel. J Physiol.

1997;499:715-720.

Nichols CG, Shyng SL, Nestorowicz A, Glaser B, Clement JP VI, Gonzalez G, Aguilar-Bryan L, Permutt MA, Bryan J. Adenosine diphosphate as an intracellular regulator of insulin secretion. Science. 1996;272:1785-1787.

Gribble FM, Tucker SJ, Ashcroft FM. The interaction of nucleotides with the tolbutamide block of cloned ATP-sensitive K+ channel currents expressed in Xenopus oocytes: a reinterpretation. J Physiol. 1997;504: 35-45.

Gribble FM, Tucker SJ, Ashcroft FM. The essential role of the Walker-A motifs of SUR1 in K-ATP channel activation by Mg-ADP and diazoxide. EMBO J. 1997;16:1145-1152.

S, Ferrigni T, Nichols CG. Regulation of KATP channel activity diazoxide and MgADP. Distinct functions of the two-nucleotide binding folds of the sulfonylurea receptor. J Gen Physiol. 1997;110: 643-654.

Shindo T, Yamada M, Isomoto S, Horio Y, Kurachi Y. SUR2 subtype (A and B)-dependent differential activation of the cloned ATP-sensitive K+ channels by pinacidil and nicorandil. Br J Pharmacol. 1998;124:985-991.

Higgins CF. The ABC of channel regulation. Cell. 1995;82:693-696. Tusnady GE, Bakos E, Varadi A, Sarkadi B. Membrane topology distinguishes a subfamily of the ATP-binding cassette (ABC) transporters.

BS Lett. 1997;402:1-3. Ueda K, Inagaki N, Seino S. MgADP antagonism to Mg2+-independent ATP binding of the sulfonylurea receptor SUR1. J Biol Chem. 1997;272: 22983-22986.

Ueda K, Matsuo M, Tanabe K, Morita K, Kioka N, Amachi T. Comparative aspects of the function and mechanism of SUR1 and MDR1 protein. Biochim Biophys Acta. 1999;1461:305-313. Reimann F, Gribble FM, Ashcroft FM. Differential response of KATP channels containing SUR2A or SUR2B subunits to nucleotides and pinacidil. Mol Pharmacol. 2000;58:1318-1325.

Matsuoka T, Matsushita K, Katayama Y, Fujita A, Inageda K, Tanemoto M, Inanobe A, Yamashita Y, Matsuzawa Y, Kurachi Y. C-terminal tails of sulfonylurea receptors control ADP-induced activation and diazoxide modulation of ATP-sensitive K+ channel Circ Res. 2000;87:873-880.

Hung L-W, Wang IX, Nikaido K, Liu P-Q, Ames G F, Kim S-H. Crystal structure of the ATP-binding subunit of an ABC transporter. Nature. 1998;396:703-707.

Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993;234:779-815. Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: a program to check the stereochemical quality of protein structures.

JApplCrys. 1993;26:283-291.

Diederichs K, Diez J, Greller G, Muller C, Breed J, Schnell C, Vonrhein C, Boos W, Welte W. Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of archaeon Thermococcus litoralis. EMBOJ. 2000;19:5951-5961.

Karpowich N, Martsinkevich O, Millen L, Yuan Y, Dai PL, MacVey K, Thomas PJ, Hunt JF. Crystal structures of the MJ1267 ATP binding cassette reveal an induced-fit effect at the ATPase active site of an ABC transporter. Structure. 2001;9:571-586.

23. Tagaya M, Yagami T, Fukui T. Affinity labeling of adenylate kinase with adenosine diphosphopyridoxal: presence of Lys21 in ATP-binding site. J Biol Chem. 1987;262:8257-8261.

24. Azzaria M, Schurr E, Gros P. Discrete mutations introduced in the predicted nucleotide binding sites of the mdr1 gene abolish its ability to confer multidrug resistance. Mol Cell Biol. 1989;9:5289-5297.

25. D'hahan N, Moreau C, Prost A-L, Jacquet H, Alekseev AE, Terzic A, Vivaudou M. Pharmacological plasticity of cardiac ATP-sensitive potassium channels toward diazoxide revealed by ADP. Proc Natl Acad Sci U S A. 1999;96:12162-12167.

26. Zingman LV, Alekseev AE, Bienengraber M, Hodgson D, Karger AB, Dzeja PP, Terzic A. Signaling in channel/enzyme multimers: ATPase transitions in SUR module gate ATP-sensitive K+ conductance. Neuron. 2001;31:233-245.

27. Matsuo M, Tanabe K, Kioka N, Amachi T, Ueda K. Different binding properties and affinities for ATP and ADP among sulfonylurea receptor subtypes, SUR1, SUR2A, and SUR2B. J Biol Chem. 2000;275:28757-28763.

28. Schwappach B, Zerangue N, Jan YN, Jan LY. Molecular basis for KATP assembly: transmembrane interactions mediate association of a K+ channel with an ABC transporter. Neuron. 2000;26:155-167.

29. Kondo C, Repunte VP, Satoh E, Yamada M, Horio Y, Matsuzawa Y, Pott L, Kurachi Y. Chimeras of Kir6.1 and Kir6.2 reveal structural elements involved in spontaneous opening and unitary conductance of the ATP-sensitive K+ channels. Receptors Channels. 1998;6:129-140.

30. Tucker SJ, Gribble FM, Proks P, Trapp S, Ryder TJ, Haug T, Reimann F, Ashcroft FM. Molecular determinants of Katp channel inhibition by ATP. EMBO J. 1998;17:3290-3296.

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Circulation Research

Journal of the American Heart Association

American

Association

Intramolecular Interaction of SUR2 Subtypes for Intracellular ADP-Induced Differential

Control of K ATP Channels

Kenji Matsushita, Kengo Kinoshita, Tetsuro Matsuoka, Akikazu Fujita, Takashi Fujikado, Yasuo

Tano, Haruki Nakamura and Yoshihisa Kurachi

Circ Res. 2002;90:554-561; originally published online February 7, 2002; doi: 10.1161/01.RES.0000012666.42782.30

Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2002 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571

The online version of this article, along with updated information and services, is located on the

World Wide Web at:

http://circres.ahajoumals.org/content/90/5/554

Data Supplement (unedited) at:

http://circres.ahajournals.org/content/suppl/2002/03/26/90.5.554.DC1

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MS#3451/R2

online figures

Online Data Supplements

Figure 1. Alignment of available NBD templates and SUR-NBDs and summary of the validation of models.

(A) Structural alignments of SUR2A-NBD2, MalK from T.litoralis,1 HisP from S.typhilium2 and MJ1267 from M.jannaschii.3 Conserved regions are boxed in red and residues corresponding to the C42 of SUR are boxed in pink.

(B) PROCHECK4 calculated the values such as the amount (%) of residues belonging to the allowed region of Ramachandran plot and predicts the resolution level of the model derived from the plot and all other stereochemical parameters including some angle torsion. In the validation of SUR2-NBD2 models using HisP structure, the amount was approximately 95%. The resolution level was assessed to be 2.0-A. All other stereochemical parameters of main chains were assessed as ' better' than mean at 2.5-A resolution (see Figure 24 for details).

(C) Structures of other nucleotide-binding proteins, MalK and MJ1267, were reported.2'3 They are now available as the templates for modeling NBDs. Using MODELLER 4,5 we also constructed the models of SUR2A-NBD2 based on the atomic coordinates of MalK and MJ1267. The sequence alignment showed that the amino acid sequence identity between SUR2A and two nucleotide-binding proteins are relatively low comparing with that of HisP 4 (MalK: 20% and MJ1267: 15%. vs. HisP: 21%). The root mean square deviations (RMSDs) among the models showed the convergence with ~3-A even for the total NBD structure. In backbone heavy atoms of the characteristic residues (Arg1344 on Walker-A and Ser1517 and Glu1518 on C42, see and Figires 4 and 6), the convergence level was ~2-A. Because these values were very low, the backbones of those residues were addressed on the same position in all three NBD2-models developed from HisP, MalK and MJ1267 structures.

Figures 2- 4. The PROCHECK results

Figures 2 and 3 are Ramachandran Plot and Main-chain parameters for C-terminal of SUR2A, respectively, while Figures 4 and 5 are those for C-terminal of SUR2B. The definition of each parameter is shown in detail in the reference.4

References

1. Diedereichs K, Diez J, Greller G, Muller C, Breed J, Schnell C, Vonrhein C, Boos W, Welte W. Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of archaeon Thermococcus

litoralis The EMBO J. 2000;19,5951-5961

2. Hung L-W, Wang IX, Nikaido K, Liu P-Q, Ames G F , Kim S-H. Crystal structure of the ATP-binding

subunit of an ABC transporter Nature. 1998;396:703-707

3. Karpowich N, Martsinkevich O, Millen L, Yuan Y, Dai PL, MacVey K, Thomas PJ, Hunt JF. Crystal

structures of the MJ1267 ATP binding cassette reveal an induced-fit effect at the ATPase active site of an ABC transporter. Structure. 2001;9:571-586

4. Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: a program to check the

stereochemical quality of protein structures. J Appl Crys. 1993;26:283-291

5. Sali A , Blundell TL. Comparative protein modelling by satisfaction of spatial

restraints. J Mol Biol. 1993;234:779-815.

Walker A

SUR2ANBD2 ---QEGEIKI HDLCVRYENN LKPVLKHVKA YIKPGQKVGI CJaRTGSGKSfe

MalKNBD ----MAGVRL VDVWKVFG— EVTAVREMSL EVKDGE FMIL L 3PSGCGKT T

HISP -----NKLHV IDLHKRYG— GHEVLKGVSL QARAGDVISI I3SSGSGKS T

MJ1267 MRDTMEILRT ENIVKYFG— EFKALDGVSI SVNKGDVTLI I

13 51 44 43 48

SUR2ANBD2 LSLAFFRMVD IFDGKIVIDG ID-------- -----ISKLP LHTLRSRLSE] 1388

MalKNBD TLRMIAGLEE PSRGQIYIGD KLVADPEK— ------GIFV PPKDRD-IAi 8 5

HISP FLRCINFLEK PSEGAIIVNG QNINLVRDKD GQLKVADKNQ LRLLRTRLT4 98

MJ1267 LINVITGFLK ADEGRVYFEN KD I TN K------------EP AELYHYGIV I 3 6

Q-loop

ILQDPILFS-IGSIRFNLDPE CKCTDD---- ---------R LWEALEIAQL 1424

MTVYDN-IAF PLKLRK---- --------VP RQEIDQRVRE 122

MTVLENVMEA PIQVLG---- --------LS KHDARERALK 136

MTVLENLLIG EINPGESPLN SLFYKKWIPK EEEMVEKAFK 136

SUR2ANBD2 MalKNBD HISP MJ1267

VFQSYALYPH VFQHFNLWSH rFQTPQPLKE

Signature motif

Walker B

SUR2 AW BD2 KNMVKSLPGG

MalKNBD VAEL----LG

HISP YLAK----VG

MJ1267 ILE F----LK

SUR 2 Ali BD 2 MalKNBD HISP MJ1267

LDATVT-EGG LTELLN-RKP IDERAQGKYP LSHLYDR-KA

LQKVVMTAFA MRAELKKLQR VLRIMQQLAE IFNHVLELKA

ENFSVGQ îQL FCLARAFVRK RELSGGy îQR VALGRAIVRK VhLSGGQ 5QR VSIARALAME GELSGGQ-IKL VEIGRALMTN

SSŒLIMDE PQwFLMDE PDft/LLFDE PKMIVMDQ

&T 1473

PL 167

PT 182

PI 181

Switch

C42 Region

-RTWTIA HR 3VTTIYVT HD2 3KTMWVT HE -1 3ITFLIIE HRL

SSIVDAG VEAMTMG ^FARHVS DIVLNYI

L-VLVFSEGI DRIAVMNRGV SHVIFLHQGK DHLYVMFNGQ

1520 217

231 230

SUR2ANBD2 MalKNBD HISP MJ1267

LVECDTGPNL LQHKNGLFST LVMTNK LQQVGSPDEV YDKPANTFVA GFIGSP IEEEGDPEQV FGNPQSPRLQ QFLKGS IIAEGRGEEE IKNVLSDPKV VEIYIGË

?MNF LDAIVTED K—--------

1546 255 2 59 2 57

PROCHECK results for SUR2-NBD2 models on HisP structure

residues in allowed regions (%) 94.9

resolution (A) -2.0

stereochemical parameter assessment better

SUR2B 95.5 -2.0

better

RMSD between models

SUR2A model on structure

identity vs RMSD vs model onHisP( A) SUR2ANBD2 1344 1517 1518 total

backbone

HisP MalK MJ1267

21 20 15

3.0 2.9

Online Data Supplements Figure 1

Ramachandran Plot

C-terminal of SUR2A

Phi (degrees)

Plot statistics

Residues in most favoured regions [A,B,L] 175 81.0%

Residues in additional allowed regions [a,b,l,p] 30 13.9%

Residues in generously allowed regions [~a,~b,~l,~p] 9 4.2%

Residues in disallowed regions 2 0.9%

Number of non-glycine and non-proline residues 216 100.0%

Number of end-residues 2

Number of glycine residues (shown as triangles) 18

Number of proline residues 8

Total number of residues 244

Based on an analysis of 118 structures of resolution of at least 2.0 Angstroms and R-factor no greater than 20%, a good quality model would be expected to have over 90% in the most favoured regions.

u T3 T3

Main-chain parameters

C-terminal of SUR2A

a. Ramachandran plot quality assessment

1.5 2.0 2.5 3.0 3.5 Resolution (Angstroms)

o T3 cö PQ

2.0 1.8 1.5 1.3 1.0 0.8 0.5 0.3 0.0

1.0 1.5 2.0 2.5 3.0 3.5 Resolution (Angstroms)

e. Hydrogen bond energies

1.0 1.5 2.0 2.5 3.0 3.5 Resolution (Angstroms)

^b. Peptide bond planarity - omega angle sd

2.0 2.5 3.0 3.5 Resolution (Angstroms)

c. Measure of bad non-bonded interactions 70-

g 17.5-

3 15.0-

d. Alpha carbon tetrahedral distortion

12.5 10.0 7.55.0 2.5 0.0

'1.0 ~L5 2!5 2.5 3.0 3T

Resolution (Angstroms)

C-terminal of SUR2A

Plot statistics

Stereochemical parameter No. of data pts Parameter value Comparison values Typical Band value width No. of band widths from mean

a. %-tage residues in A, B, L 216 81.0 76.6 10.0 0.4 Better

b. Omega angle st dev 243 3.5 6.0 3.0 -0.8 Better

c. Bad contacts / 100 residues 0 0.0 10.5 10.0 -1.1 BETTER

d. Zeta angle st dev 226 1.2 3.1 1.6 -1.2 BETTER

e. H-bond energy st dev 147 0.8 0.9 0.2 -0.6 Better

Ramachandran Plot C-terminal of SUR2B

Phi (degrees)

Plot statistics

Residues in most favoured regions [A,B,L] 178 81.7%

Residues in additional allowed regions [a,b,l,p] 30 13.8%

Residues in generously allowed regions [~a,~b,~l,~p] 6 2.8%

Residues in disallowed regions 4 1.8%

Number of non-glycine and non-proline residues 218 100.0%

Number of end-residues 2

Number of glycine residues (shown as triangles) 16

Number of proline residues 8

Total number of residues 244

Based on an analysis of 118 structures of resolution of at least 2.0 Angstroms and R-factor no greater than 20%, a good quality model would be expected to have over 90% in the most favoured regions.

Main-chain parameters

C-terminal of SUR2B

<D ■Ü

<D ■Ü

ö o o T3

100 80 60 40 20

a. Ramachandran plot quality assessment

1.0 1.5 2.0 2.5 3.0 3T Resolution (Angstroms)

60 50 40 30 20 10

2.0 1.8 1.5 1.3 1.0 0.8 0.5 0.3 0.0

1.0 1.5 2.0 2.5 3.0 3.5 Resolution (Angstroms)

e. Hydrogen bond energies

1.0 T5 2.0 2.5 3.0 3.5"

Resolution (Angstroms)

b. Peptide bond planarity - omega angle sd

c. Measure of bad non-bonded interactions

^ 20.0-s)

g 17.5. gr

3 15.0

Ü 10.0

S 7.5 ■ le

gl 5.0

1.0 1.5 2.0 2.5 3.0 3.5 4.0 Resolution (Angstroms)

d. Alpha carbon tetrahedral distortion

2.5 0.0

1.0 1.5 2.0 2.5 3.0 3.5 Resolution (Angstroms)

C-terminal of SUR2B

Plot statistics

Comparison values

No. of

Stereochemical parameter No. of data pts Parameter value Typical value Band width band widths from mean

a. %-tage residues in A, B, L 218 81.7 76.6 10.0 0.5 Better

b. Omega angle st dev 243 3.8 6.0 3.0 -0.7 Better

c. Bad contacts / 100 residues 0 0.0 10.5 10.0 -1.1 BETTER

d. Zeta angle st dev 228 1.3 3.1 1.6 -1.1 BETTER

e. H-bond energy st dev 145 0.8 0.9 0.2 -0.6 Better