Correspondence to: C.G. Nichols, Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110., cnichols{at}cellbio.wustl.edu (E-mail), Fax: 314-362-7463; (fax)
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Abstract |
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The sensitivity of KATP channels to high-affinity block by sulfonylureas and to stimulation by K+ channel openers and MgADP (PCOs) is conferred by the regulatory sulfonylurea receptor (SUR) subunit, whereas ATP inhibits the channel through interaction with the inward rectifier (Kir6.2) subunit. Phosphatidylinositol 4,5-bisphosphate (PIP2) profoundly antagonized ATP inhibition of KATP channels expressed from cloned Kir6.2+SUR1 subunits, but also abolished high affinity tolbutamide sensitivity. By stabilizing the open state of the channel, PIP2 drives the channel away from closed state(s) that are preferentially affected by high affinity tolbutamide binding, thereby producing an apparent loss of high affinity tolbutamide inhibition. Mutant KATP channels (Kir6.2[N30] or Kir6.2[L164A], coexpressed with SUR1) also displayed an "uncoupled" phenotype with no high affinity tolbutamide block and with intrinsically higher open state stability. Conversely, Kir6.2[R176A]+SUR1 channels, which have an intrinsically lower open state stability, displayed a greater high affinity fraction of tolbutamide block. In addition to antagonizing high-affinity block by tolbutamide, PIP2 also altered the stimulatory action of the PCOs, diazoxide and MgADP. With time after PIP2 application, PCO stimulation first increased, and then subsequently decreased, probably reflecting a common pathway for activation of the channel by stimulatory PCOs and PIP2. The net effect of increasing open state stability, either by PIP2 or mutagenesis, is an apparent "uncoupling" of the Kir6.2 subunit from the regulatory input of SUR1, an action that can be partially reversed by screening negative charges on the membrane with poly-L-lysine.
Key Words: K+ current, sulfonylurea, MgADP, diazoxide, PIP2
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Introduction |
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The 10 yr that followed the discovery of ATP-sensitive (KATP) channels (
Deletion of up to ~36 amino acids from the COOH terminus of Kir6.2 results in the generation of ATP-sensitive channels in the absence of SURx subunits (
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Methods |
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Molecular Biology
Kir6.2 mutations were prepared using PCR methods. Resulting PCR products were subcloned into the EcoRI-ClaI sites of the mammalian expression vector pCMV6b. SUR1 was cloned into the pECE expression vector. The nucleotide sequences of the mutant Kir6.2 constructs were verified by fluorescence-based cycle sequencing using AmpliTaq DNA polymerase, FS (Perkin-Elmer Cetus Corp.), and an ABI PRISM DNA sequencer (Perkin-Elmer Cetus Corp.).
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 fetal calf serum (10%). The next day, cells were transfected by incubation for 4 h at 37°C in DMEM containing 10% nuserum, 0.4 mg/ml diethylaminoethyl-dextran, 100 µM chloroquine, and 5 µg each of pCMV6b-Kir6.2, pECE-SUR1, and pECEgreen fluorescent protein cDNA. Cells were subsequently incubated for 2 min in HEPES-buffered salt solution containing DMSO (10%), and returned to DMEM-HG plus 10% FCS. Cells were assayed for KATP currents by patch-clamp measurements, 24 d after transfection.
Patch-Clamp Measurements
Patch-clamp experiments were made at room temperature, in an oil-gate chamber that allowed the solution bathing the exposed surface of the isolated patch to be changed rapidly. 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 (see below). Microelectrodes were "sealed" onto cells that fluoresced green under UV illumination by applying light suction to the rear of the pipette. Inside-out patches were obtained by lifting the electrode and then passing the electrode tip through the oil-gate. Membrane patches were voltage-clamped with an Axopatch 1B patch-clamp amplifier (Axon Inc.). The standard bath (intracellular) and pipette (extracellular) solution used in these experiments (K-INT) had the following composition: 140 mM KCl, 10 mM K-HEPES, 1 mM K-EGTA, pH 7.3. PIP2 was bath sonicated in ice for 30 min before use. PIP2 was obtained from Boehringer Mannheim. Tolbutamide, diazoxide, nucleotides, and poly-L-lysine (mol wt ~ 1,000) were purchased from Sigma Chemical Co. Tolbutamide and diazoxide were dissolved as stock solutions in DMSO and diluted to <1% DMSO. All currents were measured at a membrane potential of -50 mV (pipette voltage = +50 mV). Inward currents at this voltage are shown as upward deflections. Data were normally filtered at 0.53 kHz, signals were digitized at 22 kHz (Neurocorder; Neurodata) and stored on video tape. Experiments were replayed onto a chart recorder, or digitized into a microcomputer using Axotape software (Axon Inc.). Off-line 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.
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Results |
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High Affinity Sulfonylurea Sensitivity Is Lost After Kir6.2 NH2-Terminal Deletion
N2-30]+SUR1 channels also have a reduced ATP sensitivity, which in this case results from open-state stabilization that is reflected by near continuous bursting at the single channel level (
N2-30] channels because the NH2 terminus is physically involved in "coupling" to the regulatory effects of SUR1, or because the high affinity inhibitory effect of tolbutamide depends on channel open state stability.
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High Affinity Sulfonylurea Sensitivity Is Lost After PIP2 Treatment of Wild-Type Channels
We can explore the correlation between tolbutamide sensitivity and open-state stability2 of the channel by applying PIP2. PIP2 increases the channel open probability by increasing bursting behavior of the single channel and decreases the sensitivity to ATP (
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The loss of high affinity tolbutamide inhibition could occur because the high affinity component actually changes affinity (i.e., the real, or apparent, binding affinity of tolbutamide is reduced), or because high affinity binding fails to cause inhibition of channel activity. As shown in Figure 3, the latter explanation is correct; with time after addition of PIP2, the doseresponse relationship can be fit by assuming that the high affinity inhibition becomes a progressively smaller fraction of the [tolbutamide]-inhibition relationship. Data points at intermediate times cannot be fit by assuming a constant high affinity fraction, with reduced affinity. This is consistent with an effect of PIP2 on the coupling of high affinity binding to channel inhibition, not on modifying tolbutamide binding itself.
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High Affinity Sulfonylurea Sensitivity Depends on the Channel Open-State Stability
Since PIP2 and NH2-terminal deletion both increase the channel open state stability (
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MgADP Stimulation and Diazoxide Stimulation of Channel Activity Disappears with PIP2 Stimulation
Activation of wild-type Kir6.2+SUR1 channels by MgADP and diazoxide, at a fixed [ATP], is quite variable from patch to patch (Figure 5 B and 6 B). As shown in Figure 5 A and 6 A, the ability of these agents to stimulate channel activity changes after PIP2 stimulation, and in a qualitatively similar way for both Kir6.2[N2-30]+SUR1 and wild-type (Kir6.2+SUR1) channels. In each case, the stimulation tends to increase, but then gradually falls to zero with time after PIP2 application. The time course of this effect is also quite variable from patch to patch (Figure 5 B and 6 B), but is reasonably well correlated with the accompanying change of ATP sensitivity (Figure 5 D and 6 D). This result indicates that the stimulatory action of the PCOs, like ATP sensitivity itself, is not a fixed parameter of channel function, but is probably dependent on the open-state stability of the channel (
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PIP2-induced Loss of Coupling Can Be Partially Restored with Poly-l-lysine Treatment
Treatment with polycations can reverse the stimulatory actions of PIP2 on open probability and ATP sensitivity (
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Discussion |
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Loss of High Affinity Tolbutamide Sensitivity with Increased Open Probability
A biphasic doseresponse relationship for tolbutamide inhibition of KATP channels was demonstrated by N2-30] channels, which have an intrinsically higher open state stability (
The present findings are significant for understanding sulfonylurea sensitivity of KATP channels. They demonstrate that sulfonylurea sensitivity will depend critically on the open-state stability of the channels (manifested by open probability in the absence of ATP1). This can change dramatically in inside-out membrane patches as a consequence of "run down" and "run up." Run down is a gradual, variable, and probably multifactorial, reduction of channel activity, often associated with decreased open probability and increased K1/2,ATP (
PIP2 Activation Masks PCO Actions
It is clear that PIP2 activation of KATP channels and other inward rectifiers does not require the presence of a SUR1 subunit, and probably results from a direct interaction of PIP2 with the cytoplasmic portion of the channel protein itself (
The Role of SUR Subunits in Controlling KATP Channel Function
It is now clear that the pore-forming (Kir6.2) subunits can generate ATP-sensitive K channels in the complete absence of expressed SUR subunits, even without truncation of the COOH terminus (
High affinity sulfonylurea sensitivity and PCO sensitivity is conferred by the SUR1 subunit, and is absent for Kir6.2 channels expressed in the absence of SUR1 (
Conclusions
High affinity tolbutamide inhibition seems, like ATP inhibition, to be the result of a closed state stabilization, but, unlike ATP inhibition, is not likely to be a direct binding to the closed channel. Stabilizing the open state and raising the channel open probability, either by mutation or by application of PIP2, reduces high affinity tolbutamide sensitivity. Similarly, PCOs act on SUR1 to stabilize the channel in the open state, convergent with PIP2 action, such that PIP2 treatment leads to channel activation without further activation in the presence of PCOs. Treatment with polylysine causes at least partial reversal of the uncoupling actions of PIP2 effect, restoring some high affinity tolbutamide sensitivity and PCO stimulation. These results indicate that, in native cells, the pharmacological and physiological control of channel activity by the SUR1 subunit will be critically dependent on the open-state stability, itself determined by the phospholipid content of the membrane.
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Footnotes |
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2 The open state stability is the stability of the "bursting" state relative to a longer closed state that is accessible to ATP. As the open state stability increases, the open probability increases towards a saturating level of ~0.9 (i.e., the intraburst open probability), and the K1/2,ATP increases continually (
3 The increase in Po that occurs after PIP2 application is followed by a variable, very slow, loss of channel activity over many minutes ("terminal rundown"). Such rundown occurs in the presence or absence of PIP2. This terminal rundown may occur by channels terminally disappearing from the patch, the open probability estimated by noise analysis (i.e., the open probability of channels that remain functional) does not decline during this process, as quantified for the record in Figure 6 A.
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Acknowledgements |
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We thank Jeremiah Shepard and Rebecca Dunlap for technical assistance with these experiments. We are grateful to Dr. S. Seino for providing us with the Kir6.2 clone.
This work was supported by a grant from the National Institutes of Health (NIH) (HL45742 to C.G. Nichols), a Career Development Award from the American Diabetes Association (S.-L. Shyng, the Washington University Diabetes Research and Training Center (NIH Training grant support of J.C. Koster, and reagents), and the Washington University Cardiovascular NIH Training Grant (fellowship support for Q. Sha).
Submitted: February 3, 1999; Revised: June 17, 1999; Accepted: June 18, 1999.
1used in this paper: Kir, inward rectifier; PCO, potassium channel opener; PIP2, phosphatidylinositol 4,5-bisphosphate; SUR, sulfonylurea receptor
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