Correspondence to: Colin G. Nichols, Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110. Fax:(314) 3620-7463 E-mail:cnichols{at}cellbio.wustl.edu.
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Abstract |
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Phosphatidylinositol 4,5-bisphosphate (PIP2) activates KATP and other inward rectifier (Kir) channels. To determine residues important for PIP2 regulation, we have systematically mutated each positive charge in the COOH terminus of Kir6.2 to alanine. The effects of these mutations on channel function were examined using 86Rb efflux assays on intact cells and inside-out patch-clamp methods. Both methods identify essentially the same basic residues in two narrow regions (176222 and 301314) in the COOH terminus that are important for the maintenance of channel function and interaction with PIP2. Only one residue (R201A) simultaneously affected ATP and PIP2 sensitivity, which is consistent with the notion that these ligands, while functionally competitive, are unlikely to bind to identical sites. Strikingly, none of 13 basic residues in the terminal portion (residues 315390) of the COOH terminus affected channel function when neutralized. The data help to define the structural requirements for PIP2 sensitivity of KATP channels. Moreover, the regions and residues defined in this study parallel those uncovered in recent studies of PIP2 sensitivity in other inward rectifier channels, indicating a common structural basis for PIP2 regulation.
Key Words: potassium channel, ATP, PH domain, Kir6.2, phospholipid
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INTRODUCTION |
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ATP-sensitive potassium (KATP)1 channels couple cell metabolism to excitability in many tissues (C]) enables the formation of functional channels in the absence of SUR (
ATP inhibits KATP channels with half-maximal inhibitory concentration (K1/2,ATP) of 10 µM in excised patches (
The antagonistic effect of PIP2 on ATP inhibition suggests that the two ligands compete functionally for interaction with the channel. The Kir6.2 subunit is primarily responsible for these interactions, since Kir6.2[C] expressed without SUR1 (
We have now performed systematic mutagenesis and electrophysiological analysis to determine the positively charged residues in the Kir6.2 COOH terminus that are critical for PIP2 interaction. We report multiple such residues clustered in the proximal COOH terminus, and consider the possible structural basis for channel regulation by phospholipids.
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MATERIALS AND METHODS |
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Molecular Biology
Constructs containing point mutations were prepared by overlap extension at the junctions of the relevant residues by sequential PCR. The resulting PCR products were subcloned into the pCMV6b vector. Before transfection, the constructs were sequenced to verify the correct mutations.
Expression of KATP channels in COSm6 Cells
COSm6 cells were plated at a density of 2.5 x 105 cells per well (30-mm six-well dishes) and cultured in Dulbecco's Modified Eagle Medium plus 10 mM glucose (DMEM-HG) supplemented with 10% FCS. The next day, cells were transfected by incubation for 4 h at 37°C in DMEM medium containing 10% Nuserum, 0.4 mg/ml diethylaminoethyl-dextran, 100 µM chloroquine, and 5 µg each of pCMV6b-Kir6.2 or mutant isoforms, pECE-SUR1 cDNA, and pECE-GFP (green fluorescent protein). Cells were subsequently incubated for 2 min in HEPES-buffered salt solution containing 10% DMSO, and returned to DMEM-HG plus 10% FCS.
86Rb+ Efflux Assay
Cells were incubated for 24 h in culture medium containing 86RbCl (1 µCi/ml) for 23 d after transfection. Before measurement of Rb efflux, cells were incubated for 30 min at 25°C in Krebs' Ringer solution, with metabolic inhibitors (2.5 µg/ml oligomycin plus 1mM 2-deoxy-D-glucose). At selected time points, the solution was aspirated from the cells and replaced with fresh solution. At the end of the 40-min period, cells were lysed in 2% SDS-Ringer's solution. The 86Rb+ in the aspirated solution and the cell lysates were counted. The percent efflux at each time point was calculated as the cumulative counts in the aspirated solution divided by the total counts from the solutions and the cell lysates.
Patch-Clamp Measurements
Patch-clamp experiments were made at room temperature in a chamber that allowed rapid exchange of bathing solution. Micropipettes were pulled from thin-walled glass (WPI Inc.) on a horizontal puller (Sutter Instrument, Co.). Electrode resistance was typically 0.51 M when filled with K-INT solution (below). Inside-out patches were voltage-clamped with an Axopatch 1B amplifier (Axon Inc.). The standard bath (intracellular) and pipette (extracellular) solution (K-INT) had the following composition: 140 mM KCl, 10 mM K-HEPES, and 1 mM K-EGTA, pH 7.3. PIP2 was bath-sonicated in ice for 30 min before use. ATP was added as the potassium salt. All currents were measured at a membrane potential of -50 mV (pipette voltage = +50 mV), and inward currents at this voltage are shown as upward deflections. Data were filtered at 0.53 kHz, digitized at 22 kHz (Neurocorder; Neurodata), and stored on videotape. Experiments were replayed onto a chart recorder or digitized into a microcomputer using Axotape software (Axon Inc.). Offline analysis was performed using Microsoft Excel programs. Wherever possible, data are presented as mean ± SEM. Microsoft Solver was used to fit data by a least-square algorithm.
Interpretation of PIP2 Response Data
Wild-type KATP (Kir6.2 + SUR1) channels have an intrinsic open probability in the absence of ATP (Po,zero) of 0.4 and are inhibited by ATP with K1/2,ATP of
10 µM (
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RESULTS |
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Residues Critical for Regulation of Kir6.2 by PIP2
In the Kir6.2 COOH terminus (residues 173390), there are 23 basic residues (Fig 1 A), any or all of which might contribute electrostatically to PIP2 binding. We performed alanine scanning mutagenesis of these basic residues to determine their involvement in PIP2 sensitivity and/or ATP sensitivity. Channel activity of all mutants was initially assessed using a 86Rb+ efflux assay (Fig 1 B). In addition to the previously recognized R176 and R177 residues (
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Mutant channel activity was examined in more detail in inside-out membrane patches. Wild-type Kir6.2 + SUR1 channels have an intrinsic Po,zero of 0.4 (
0.9 (Fig 2A and Fig C). As considered above, mutations that reduce apparent PIP2 affinity, either by real changes in PIP2 binding affinity or by lowering the intrinsic stability of the open pore, will lower the intrinsic Po,zero. When membrane PIP2 is increased by cytoplasmic exposure, the increase in current in these mutants should occur more slowly than wild-type, and to a relatively greater extent.2 The sensitivity of mutant channels to PIP2 stimulation was estimated from the time course (Fig 2 B) and the extent (Fig 2 C) of increase in relative current in response to cytoplasmic application of 5 µg/ml PIP2. Nine mutations were identified as having altered sensitivity to PIP2 in this assay (R176A, R177A, R195A, R206A, K222A, R301A, and R314A [identified above], plus R192A and R201A, which also show a nonsignificant reduction in Rb flux compared with wild-type). Again, there was no apparent effect of mutations downstream of R314 on PIP2 sensitivity. The correlation between mutant effects on Rb efflux and response to PIP2 indicates that, in each case, reduction in Rb efflux is likely due to a reduced sensitivity to the ambient phospholipid level in the intact cell.
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As shown previously (1 mM (
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Nonidentical Residues Control ATP and PIP2 Sensitivity
All expressed mutants had comparable intrinsic ATP sensitivity to wild-type channels (Fig 2 D) except K185Q and R201A. After PIP2 stimulation, K1/2,ATP increased to between 1 and 10 mM for all mutants. Of the PIP2-sensitive residues, only R201A (Fig 4) also affects sensitivity to ATP. Compared with wild-type channels, R201A mutant channels actually showed only moderately increased stimulation by PIP2 (Fig 2) and no significant reduction of 86Rb efflux in intact cells (Fig 1 B). However, R201A channels showed a significantly decreased ATP sensitivity (Ki 115 µM; Fig 4 B). Since the mutation reduces ATP sensitivity without increasing Po,zero, R201 is a candidate ATP binding site residue. A change in ATP binding affinity might also result in altered cooperativity between subunits, which could be an explanation for the experimentally significant reduction of the Hill coefficient (H) for inhibition by ATP (Fig 4 B). The shift of ATP sensitivity in this mutation is similar to that observed for the K185Q mutant (Fig 2 D), which has lower affinity for ATP binding (
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Like R177A, the R206A and K222A mutant channels displayed no activity after membrane excision. There was no detectable response of either mutant to a 10-min exposure to 5 µg/ml PIP2 (not shown), but R206A channel activity did appear after several minutes of exposure to a much higher concentration of PIP2 (100 µg/ml; Fig 4 C), indicating that the lack of current after patch excision reflects a much lower open state stability. The K222A mutant showed no activity even on exposure to 100 µg/ml PIP2. However, like the R177A mutant, K222A channels could be rescued by coexpression with L157C mutants (Fig 3 B), generating channels that were much more sensitive to ATP (Fig 3 B; K1/2,ATP = 0.085 mM) than L157C + SUR1 alone. Again, this result indicates that the lack of current in the K222A mutant results from channels being closed, which is consistent with reduced PIP2 affinity, and not from any gross structural defect.
Some Mutants Display Prominent PIP2-sensitive Inactivation
Interestingly, three of the six mutations (R192A, R301A, and R314A) showed inactivation after removal of ATP, and this inactivation was most pronounced in the R301A mutant (Fig 5). After patch excision, the estimated time constant of inactivation, after a step from 1 mM ATP to zero ATP, was 10.9 ± 0.5 s for R192A, 2.7 ± 0.3 s for R301A, and 19.5 ± 4.7 s for R314A (n = 410 patches). We previously observed similar inactivation in an M2 pore mutation (N160Q; 85 and
70% of wild-type, respectively (Fig 1 B). Given the stimulatory effect of MgADP in intact cells, and that PIP2 reverses the inactivation process, it is conceivable that these agents sustain activity of the less severe inactivation mutants (R192A and R314A) in intact cells.
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DISCUSSION |
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Distinct Residues Are Involved in Channel Gating of Kir6.2 Channels by ATP and PIP2
The mechanism of PIP2 activation of inward rectifier channels, and of ATP inhibition of KATP channels, remain elusive. It has been suggested previously that electrostatic interaction of the Kir subunit cytoplasmic domain with phospholipids in the membrane stabilizes the open state of the channel (
Regulation of Other Inwardly Rectifying Potassium Channels by Membrane PIP2
Other inward rectifier channels in the Kir family are regulated by membrane PIP2 (
The regions equivalent to residues 206222 in Kir6.2 have been mutated in several studies on various Kir channels. E224 in Kir2.1 (S212 in Kir6.2) is a major determinant of the affinity for pore-blocking polyamines (
Critical residues involved in PIP2 regulation are conserved, or appropriate changes in PIP2 sensitivity are observed, when such residues are introduced to different Kir channels (
The present study was undertaken without any knowledge, or presupposition, of the overall structure of the cytoplasmic domain. In the absence of any homology to known structures, we performed a prediction of the secondary structure of the COOH terminus of Kir channels using multiple alignments of the primary sequences (PHD program [ 150 amino acids of the COOH termini of all Kir channels are likely to contain seven antiparallel ß-strands with an
-helix at the COOH-terminal end. These are characteristics of pleckstrin homology (PH) domains (
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Footnotes |
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1 Abbreviations used in this paper: KATP, ATP-sensitive potassium; PH, pleckstrin homology; PIP2, phosphatidylinositol 4,5-bisphosphate; SUR, sulfonylurea receptor.
2 Wild-type channels have a Po,zero of 0.45 under normal conditions, and this rises to a maximum of
0.9 after addition of PIP2, so the macroscopic relative current approximately doubles. Mutations that reduce the apparent PIP2 affinity will reduce the ambient Po,zero, and this means that the potential increase of Po,zero, after addition of PIP2, is greater. However, unless PIP2 efficacy is reduced, or the affinity is reduced so far that it becomes impossible to add sufficient PIP2 (as may in fact be the case for R206A), Po,zero would still rise eventually to the same saturating value (
0.9). Hence, the increase in the relative current will be greater, but take longer, than in wild-type channels.
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Acknowledgements |
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We are grateful to the Washington University Diabetes Research and Training Center for continued molecular biology supplies.
This work was supported by a career development grant from the American Diabetes Association (to S.L. Shyng) and grants HL45742 and HL54171 from the National Institutes of Health (to C.G. Nichols).
Submitted: 21 June 2000
Revised: 11 September 2000
Accepted: 11 September 2000
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