Modulation of nucleotide sensitivity of ATP-sensitive potassium channels by phosphatidylinositol-4-phosphate 5-kinase

Abstract

ATP-sensitive potassium channels (K_ATP channels) regulate cell excitability in response to metabolic changes. K_ATP channels are formed as a complex of a sulfonylurea receptor (SURx), a member of the ATP-binding cassette protein family, and an inward rectifier K⁺ channel subunit (Kir6.x). Membrane phospholipids, in particular phosphatidylinositol (PI) 4,5-bisphosphate (PIP₂), activate K_ATP channels and antagonize ATP inhibition of K_ATP channels when applied to inside-out membrane patches. To examine the physiological relevance of this regulatory mechanism, we manipulated membrane PIP₂ levels by expressing either the wild-type or an inactive form of PI-4-phosphate 5-kinase (PIP5K) in COSm6 cells and examined the ATP sensitivity of coexpressed K_ATP channels. Channels from cells expressing the wild-type PIP5K have a 6-fold lower ATP sensitivity (K_1/2, the half maximal inhibitory concentration, ≈ 60 μM) than the sensitivities from control cells (K_1/2 ≈ 10 μM). An inactive form of the PIP5K had little effect on the K_1/2 of wild-type channels but increased the ATP-sensitivity of a mutant K_ATP channel that has an intrinsically lower ATP sensitivity (from K_1/2 ≈ 450 μM to K_1/2 ≈ 100 μM), suggesting a decrease in membrane PIP₂ levels as a consequence of a dominant-negative effect of the inactive PIP5K. These results show that PIP5K activity, which regulates PIP₂ and PI-3,4,5-P₃ levels, is a significant determinant of the physiological nucleotide sensitivity of K_ATP channels.

The pancreatic ATP-sensitive potassium channel (K_ATP channel) is formed by association of a sulfonylurea receptor (1) with an inward rectifier K channel subunit (2–6). K_ATP channels serve as molecular sensors of blood glucose levels to regulate insulin secretion in pancreatic β-cells by coupling cell metabolism to cell excitability. The hallmark feature of K_ATP channels is their inhibition by intracellular ATP, with half-maximal inhibitory concentration (K_1/2) measured by inside-out patch-clamp techniques being ≈10 μM (7, 8). Additionally, in the presence of Mg²⁺, ADP antagonizes the inhibitory effect of ATP and stimulates channel activity (7, 9). It has long been recognized that activation of K_ATP channels occurs under conditions where the cytoplasmic ATP is much higher than that required to inhibit channels in excised membrane patches (7, 10), and accumulating evidence suggests that in vivo activation of K_ATP channels is, at least in part, due to ADP stimulation of channels after glucose starvation (11). Hilgemann and Ball (12) and Fan and Makielski (13) showed that application of phosphoinositides (PIPs), in particular phosphatidylinositol (PI) 4,5-bisphosphate (PIP₂), to inside-out membrane patches activates K_ATP channels. We recently showed that PIP₂ dramatically reduced the sensitivity of K_ATP channels to inhibition by ATP (14, 15). These observations raise the possibility that, in addition to ADP, membrane PIPs may play a key role in physiological activation of K_ATP channels.

PIPs are phosphorylated derivatives of PI. The PIPs that have been detected in cells include PI-3-P, PI-4-P (PIP), PI-5-P, PI-3,4-P₂, PI-4,5-P₂ (PIP₂), PI-3,5-P₂, and PI-3,4,5-P₃ (PIP₃). PIP and PIP₂ are the most abundant forms, comprising ≈60% of total PIPs (16, 17). Comparing the potency of PIP, PIP₂, and PIP₃ in their ability to modulate the nucleotide sensitivity of K_ATP channels, we find that PIP₂ is much more effective than PIP and about equally effective as PIP₃ (14). To examine whether endogenous membrane PIP₂ and/or PIP₃ can indeed regulate K_ATP channel activity, we perturbed the levels of these PIPs by manipulating the activity of a lipid kinase involved in their synthesis. Phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) are enzymes involved in the synthesis of PIP₂, PI-3,5-P₂, and PIP₃. Two isoforms of PIP5Ks were characterized from erythrocytes; these were denoted type I and type II PIP5K based on their biochemical properties (18–20). In mammalian cells, three isoforms of PIP5Ks (α, β, and γ) have been cloned (21–24). Although these isoforms share a marked sequence homology in their central portion (the putative kinase catalytic domain), the N- and C-terminal regions differ significantly. Little is known about the cellular distribution of the various PIP5K isoforms or the cellular processes that are regulated by these kinases (17, 25). In a search for proteins that complement growth factor receptor-dependent mitogenic signaling, Davis et al. (26) isolated a truncated form of the murine type I PIP5K β-isoform that lacks the N-terminal 238 amino acids (PIP5K:Δ1–238) and is inactive in converting PIP to PIP₂. The authors showed that overexpression of PIP5K:Δ1–238 resulted in stabilization of a growth factor receptor that normally undergoes endocytosis from the cell surface on ligand stimulation, suggesting a role of PIP5K in receptor endocytosis, possibly by regulating the level of PIP₂ and/or PIP₃ in the membrane.

In the present study, we overexpressed the wild-type type Iβ PIP5K (PIP5K:WT) or the N-terminally deleted mutant kinase PIP5K:Δ1–238 and examined the effects of these manipulations on nucleotide sensitivity of K_ATP channels. We report that PIP5K activity plays an important role in determining the ATP sensitivity of these channels.

Materials and Methods

Molecular Biology.

Constructs containing point mutations were prepared by overlap extension at the junctions of the relevant residues by sequential PCR. Resulting PCR products were subcloned into pECE or pCMV6b vector and sequenced to verify the correct mutant construct before transfection.

Expression of K_ATP Channels in COSm6 Cells.

COSm6 cells were plated at a density of ≈2.5 × 10⁵ cells per well (30-mm six-well dishes) and cultured in DMEM plus 10 mM glucose supplemented with 10% (vol/vol) FCS. The next day, cells were transfected by incubation for 4 h at 37°C in DMEM containing 10% (vol/vol) Nuserum, 0.4 mg/ml DEAE-dextran, 100 μM chloroquine, and 5 μg each of pCMV6b-Kir6.2 or mutant isoforms and pECE-SUR1 cDNA. Cells were subsequently incubated for 2 min in Hepes-buffered salt solution containing 10% (vol/vol) DMSO and returned to DMEM plus 10 mM glucose and 10% (vol/vol) FCS. Cells were assayed for K_ATP currents by inside-out patch-clamp measurements 2–4 days after transfection.

Construction of Recombinant Sindbis Viruses Expressing the PIP5K:WT and PIP5K:Δ1–238 Mutant.

The two cDNAs were amplified from the pCDNA3 GFP-PIP5K:WT construct (GFP, green fluorescent protein; ref. 26) by PCR (25 cycles). The primers used had a 5′ XbaI linker followed by, first, 15 nucleotides for 5′ PIP5K:WT; second, 24 nucleotides for 3′ PIP5K:WT; and third, 26 nucleotides for the Δ1–238 mutant. The PCR products were subcloned first in the Sindbis shuttle vector (pH2J1; ref. 27). After the correct sequences and orientation of the inserts were confirmed by DNA sequencing, the inserts were subcloned (ApaI and XhoI sites) into the Sindbis virus vector (pTOTO3′2J). RNA transcripts were produced in vitro from pTOTO3′2J PIP5K:WT and PIP5K:Δ1–238 constructs with the SP6-DNA-dependent RNA polymerase. Recombinant viruses were generated from these transcripts for transfection of BHK cells. Viruses were harvested 24 h after transfection and titered (≈10⁸ plaque-forming units/ml) on BHK monolayers.

Preparation of Membrane and Cytosolic Fractions from Cells Expressing GFP-PIP5K:WT and GFP-PIP5K:Δ1–238.

Cells transfected with either PIP5K:WT or PIP5K:Δ1–238 were used to prepare membrane and cytosolic fractions as described (28). Briefly, the cells were homogenized in 20 mM Hepes-NaCl, pH 7.0/0.25 M Sucrose/0.5 mM EGTA and pelleted for 5 min at 600 × g to eliminate nuclei and intact cells. The supernatant was centrifuged for 15 min at 50,000 × g. The pellet (membrane fraction) was resuspended in homogenization buffer containing 1% Triton X-100, 0.5 μM para-PMSF, and 1 μM leupeptin and then frozen in liquid nitrogen and stored at −80°C. Aliquots were used to determine the distribution of GFP-PIP5K:WT and GFP-PIP5K:Δ1–238 mutant by Western blotting by using anti-GFP antibodies (polyclonal antisera from CLONTECH).

Expression of PIP5K in COSm6 Cells.

COSm6 cells transfected with K_ATP channel protein subunits were subsequently infected with recombinant Sindbis viruses expressing either the wild-type or the mutant PIP5K. High multiplicity of infection (50 plaque-forming units per cell) was employed to ensure high levels of infection. Virus adsorption was performed at 4°C for 1 h with rocking. Cells were incubated in regular medium for another 10–16 h at 37°C before being used for patch-clamp experiments.

Measurement of PIP Levels.

COSm6 cells were plated in 35-mm dishes. At 2 days before use, the medium was aspirated and replaced with inositol-free DMEM containing 3% (vol/vol) FCS and 1 μCi/ml [³H]myo-inositol. Cells were infected with Sindbis viruses expressing various PIP5K isoforms 12 h before the experiment. To measure PIPs, cells were washed in cold PBS, scraped into 1 ml of methanol:concentrated HCl (10:1, vol/vol). One ml of water was added, and the samples were extracted with 2 ml of chloroform. The upper, aqueous layer was removed, and the organic layer was reextracted with methanol:1 M HCl (1:1, vol/vol). Samples were evaporated to dryness, and PIPs were separated on thin layer plates as described (29). Plates were sprayed with En3Hance (NEN) and exposed to x-ray film. PI, PIP, and PIP₂ were identified by comigration with standards. Bands corresponding to these lipids were scraped from the plates and counted for ³H.

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 in less than 50 ms. Micropipettes were pulled from thin-walled glass (WPI Instruments, New Haven, CT) on a horizontal puller (Sutter Instruments, Novato, CA). Electrode resistance was typically 0.5–1 MΩ when filled with K-INT solution (see below). Microelectrodes were “sealed” by applying light suction to the rear of the pipette to cells that fluoresced green under UV illumination. 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 (Axon Instruments, Foster City, CA). 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. 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 0.5–3 kHz; signals were digitized at 22 kHz (Neurocorder, Neurodata, New York) and stored on video tape. Experiments were replayed onto a chart recorder or digitized into a microcomputer by using axotape software (Axon Instruments). Off-line analysis was performed with microsoft excel. Wherever possible, data are presented as means ± SEM. microsoft solver was used to fit ATP-dose response curves by a least-square algorithm. Goodness of fit for the individual fits was tested with the χ² test of goodness of fit. The χ² value of all curves is <χ_0.05². Test of significance was performed by Student's unpaired t test. P values of <0.05 were considered significantly different.

Results

Expression Level of PIP5K:WT and PIP5K:Δ1–238.

To monitor the expression of PIP5K, we used viral constructs with the GFP attached to the N terminus of PIP5K (Fig. 1). At 10 h after infection, more than 90% of COSm6 cells were green (data not shown). Western blot analysis with anti-GFP antibodies showed that PIP5K:WT and PIP5K:Δ1–238 were expressed at a similar level (Fig. 1), although, interestingly, the subcellular distribution of PIP5K:WT is different from that of PIP5K:Δ1–238. Although the majority of the wild-type kinase is found in the membrane fraction, most of the Δ1–238 kinase is in the cytosolic fraction.

Figure 1.

Expression and distribution of the PIP5K:WT and PIP5K:Δ1–238 mutant. (A) Cells were transfected with Sindbis virus expressing GFP-PIP5K:WT or GFP-PIP5K:Δ1–238 for different times as indicated, and PIP5K proteins were analyzed by Western blotting with anti-GFP antibodies. (B) Distribution of PIP5K:WT and PIP5K:Δ1–238 mutant in membrane and cytosol. After infection, cytosolic (cy) and membrane (mb) fractions were prepared as described in Materials and Methods, and the distributions of PIP5K protein were analyzed by Western blotting with anti-GFP antibodies.

Overexpression of PIP5K:WT Decreases ATP Sensitivity of K_ATP Channels.

The ATP-sensitivity of K_ATP channels was measured 10–16 h after viral infection by rapid application of solutions of different ATP concentrations, after excision of inside-out membrane patches. The activity of K_ATP channels in membrane patches typically decreases within a few minutes, a phenomenon known as channel “rundown.” This phenomenon has been attributed to a decrease in the levels of membrane PIPs (12, 13). To minimize time-dependent changes of PIP concentrations in the membrane patch, which could obscure our results, the ATP-sensitivity was measured immediately (within 1 min) after patch excision. Patches that showed significant channel rundown within the first minute were excluded from the analysis. Representative current traces recorded from control cells and cells infected with PIP5K:WT or with PIP5K:Δ1–238 are shown in Fig. 2A. The dose-response relationships of channel inhibition by ATP were fit by the Hill equation {I_rel = 1/[1 + ([ATP]/K_1/2)^H]; I_rel = current in [ATP]/current in zero ATP} to averaged data. For channels from control cells, the K_1/2 ([ATP] causing half-maximal inhibition) is 8 μM, with a Hill coefficient (H) of 1.3 (n = 26), similar to the K_1/2 values reported previously under identical conditions (30). For cells overexpressing PIP5K:WT, the K_1/2 increased by ≈6-fold to a value of 59 μM (n = 15; Fig. 2), significantly higher than that seen in control cells (P < 0.05). Channels in cells infected with a control virus that does not carry the PIP5K:WT gene have ATP sensitivity similar to the sensitivities in uninfected cells (data not shown).

Figure 2.

Overexpression of PIP5K:WT decreases the sensitivity of K_ATP channels to inhibition by ATP. (A) Representative wild-type K_ATP channel currents recorded in inside-out membrane patches from control cells, cells overexpressing PIP5K:WT (WT Kinase), or cells overexpressing PIP5K:Δ1–238 (ΔN Kinase). Currents were recorded at −50 mV and are shown as upward deflections. The patch was exposed to differing [ATP] as indicated by the bars above the record. (B) K_1/2 estimated for wild-type K_ATP channels in cells overexpressing PIP5K:WT or cells overexpressing PIP5K:Δ1–238, from fits of the Hill equation {I_rel = 1/[1 + ([ATP]/K_1/2)^H]}, with I_rel being the current in ATP, relative to the current in the absence of ATP. The Hill coefficient H was fixed at 1.3. Each data point represents the average of 15–26 patches. The error bar is the SEM. (Inset) PIP₂ levels measured in control cells, cells overexpressing PIP5K:WT, or cells overexpressing PIP5K:Δ1–238. Results represent the means ± SD of triplicate determinations.

Overexpression of PIP5K:Δ1–238 Increases the ATP Sensitivity of a Mutant K_ATP Channel.

The PIP5K:Δ1–238 mutant lacks kinase activity (26). In addition, it functions as a dominant-negative mutant and blocks the activity of the wild-type PIP5K (J.N.D., unpublished work; A.B. and P.D.S., unpublished work). Overexpression of PIP5K:Δ1–238 is therefore expected to lower the PIP₂ levels in the membrane. Consequently, the K_ATP channels are expected to have a decreased open state stability. Based on experimental data collected from several studies, a positive but nonlinear correlation exists between the channel open probability in the absence of ATP (P_{o zero}) and ATP-sensitivity (K_1/2) (D. Enkvetchakul, G. Loussouarn, E. Makhina, S.-L.S., and G.G.N., unpublished work). As open state stability increases above that of wild-type channels, P_{o zero} saturates, and the K_{1/2, ATP} rises. As open state stability falls toward and below that of the wild-type channel (P_{o zero} ≈ 0.4), ATP-sensitivity approaches a minimum (ref. 4; D. Enkvetchakul, G. Loussouarn, E. Makhina, S.-L.S., and G.G.N., unpublished work). Consistent with these observations, we have previously shown that raising the PIP₂ in the membrane (or making mutations that intrinsically stabilize the open state of the channel) resulted in reduced ATP-sensitivity. Conversely, lowering the level of accessible PIP₂ by treatment with poly-l-lysine or introducing mutations that reduce PIP₂ binding (as for the Kir6.2 mutation Arg-176 to Ala) did not increase ATP-sensitivity beyond that of the wild-type channel (14). Accordingly, expression of PIP5K:Δ1–238 had no effect on the ATP-sensitivity of wild-type K_ATP channels (K_1/2 = 11 μM; n = 16; Fig. 2B). To determine whether overexpression of PIP5K:Δ1–238 did decrease membrane PIP₂ levels and had an effect on K_ATP channel activity, we made use of a mutant K_ATP channel (Kir6.2[I154C, C166S] + SUR1) that has a lower ATP sensitivity than that of the wild-type channels because of an increased intrinsic open state stability (D. Enkvetchakul, G. Loussouarn, E. Makhina, S.-L.S., and G.G.N., unpublished work). Because the ATP-sensitivity is a better indicator of higher channel open state stabilities, the mutant channel is expected to be a better indicator of reductions in PIP₂ levels. In control uninfected cells, this mutant channel has a K_1/2 of 447 μM (Fig. 3). In cells infected with the PIP5K:Δ1–238 virus, ATP sensitivity of the mutant channel increased by ≈5 fold, with a K_1/2 of 97 μM (Fig. 3B), significantly lower than that seen in control uninfected cells (P < 0.05). These results are consistent with the expression of PIP5K:Δ1–238 actually resulting in a decrease in the membrane PIP₂ level. To confirm that overexpression of PIP5K:WT results in an increase in membrane PIP₂ levels, whereas overexpression of PIP5K:Δ1–238 results in a decrease, we directly measured the amount of cellular PIP₂. Whereas cells infected with wild-type kinase (PIP5K:WT) show modest elevation of total PIP₂ levels compared with control cells, cells infected with the dominant-negative construct (PIP5K:Δ1–238) show reduced total PIP₂ levels (Fig. 2B Inset). Because there is no available assay for surface membrane PIP₂ levels, these data provide a lower-limit estimate of PIP₂ levels in the surface membrane. The left shift in ATP sensitivity of a mutant K_ATP channel (Kir6.2[I154C, C166S] + SUR1) coexpressed with kinase lacking enzymatic activity can be restored by subsequent addition of exogenous PIP₂. In fact, the ATP-sensitivity can be right shifted to levels well beyond those seen in control cells (Fig. 3B Inset).

Figure 3.

Overexpression of PIP5K:Δ1–238 increases the sensitivity of a mutant K_ATP channel (Kir6.2[I154C, C166S] + SUR1) to inhibition by ATP. (A) Representative (Kir6.2[I154C, C166S] + SUR1) K_ATP channel currents recorded in inside-out membrane patches from control cells or cells overexpressing PIP5K:Δ1–238 (ΔN Kinase). Currents were recorded at −50 mV and are shown as upward deflections. The patch was isolated and exposed to differing [ATP] as indicated by the bars above the record. The dashed line indicates zero current. (B) To estimate the K_1/2 in control cells or cells overexpressing PIP5K:Δ1–238, data points were fitted to the Hill equation {I_rel = 1/[1 + ([ATP]/K_1/2)^H]}, with I_rel being the current relative to the current in the absence of ATP and with H being fixed at 1.3. Each data point represents the average of 4–6 patches, with the error bar being the SEM. (Inset) Response of (Kir6.2[I154C, C166S] + SUR1) K_ATP channels in an isolated inside-out membrane patch from cells overexpressing PIP5K:Δ1–238 to exogenous PIP₂. Currents were recorded at −50 mV and are shown as upward deflections. The patch was exposed to differing [ATP] and 5 μg/ml PIP₂ as indicated by the bars above the record. The K_1/2 increased from 219 μM to >5 mM with continued PIP₂ application.

Discussion

ATP inhibition is the hallmark feature of K_ATP channels (31). The finding that the ATP sensitivity of K_ATP channels can be modulated over orders of magnitude by exogenous application of PIP₂ on the cytoplasmic side of membrane patches (14, 15) suggests the important possibility that the ATP sensitivity of K_ATP channels is not a fixed physiological parameter but a parameter controlled by the PIPs in the plasma membrane. The physiological relevance of K_ATP channel regulation by PIPs has been implicated by our demonstration that a mutant K_ATP channel (Kir6.2 Arg-176 to Ala), which is predicted to have a lower affinity for PIP₂ binding, has very low activity in intact cells (14). Using a Xenopus oocyte expression system, Baukrowitz et al. (15) showed that activation of the P2Y₂ receptor, which activates phospholipase C and thus decreases PIP₂ concentration, reduced K_ATP channel activity and rendered channels more sensitive to ATP. The present study shows that the sensitivity of K_ATP channels to ATP inhibition can be modulated over an order of magnitude in either direction through direct manipulation of the activity of a lipid kinase that is responsible for the synthesis of PIP₂ and PIP₃. These results provide direct evidence for the hypothesis that PIPs play an important role in the regulation of K_ATP channel activity in intact cells.

K_ATP Channels as Molecular Indicators of the PIP₂/PIP₃ Levels in the Plasma Membrane.

Assuming sufficient supplies of the substrates, overexpression of PIP5K:WT is expected to increase the levels of PIP₂, whereas the expression of PIP5K:Δ1–238 is expected to decrease the levels of PIP₂. Overexpression of PIP5K:WT or PIP5K:Δ1–238 is correlated with an increase or decrease in the cellular PIP₂ levels, respectively. Because we are measuring total cellular PIP₂ levels, it is not clear how many of these changes occur at the plasma membrane where the channels are located. The present results suggest that K_ATP channels might in fact be useful as bioindicators of plasma membrane PIP₂/PIP₃.

Overexpression of PIP5K:Δ1–238 caused a decrease in the membrane PIP₂ or PIP₃ levels and increased the ATP sensitivity of a mutant K_ATP channel with a low intrinsic ATP sensitivity. How the PIP5K:Δ1–238 exerts a dominant-negative effect on the PIP5K:WT activity to lower PIP₂/PIP₃ levels is not yet clear. It is possible that the PIP5K:Δ1–238 competes for the biosynthesis of the endogenous wild-type PIP5K. It is also possible that wild-type kinases exist as multimers, and the truncated mutant kinase interferes with the normal function of the wild-type PIP5K by competing with the wild-type PIP5K for incorporation into the multimers. The fact that most PIP5K:Δ1–238 is found in the cytosolic fraction, whereas the wild-type kinase is found in the membrane fraction (Fig. 1), suggests the possibility that association of the mutant kinase with the wild-type kinase may lead to mislocalization of the enzyme. No studies have yet been performed to examine the likelihood of multimeric assembly of lipid kinases, but the present results suggest that this distinct possibility warrants investigation.

The Roles of Other Enzymes in the PIP Metabolic Pathway in Regulating K_ATP Channel Activity.

The metabolic pathways of cellular PIPs are very complex (16, 17). The amount of each PIP is dynamically regulated by the various lipid kinases, lipid phosphatases, and phospholipases. To have a comprehensive understanding of how K_ATP channel activity is regulated by PIPs, the role of individual kinase, phosphatase, and lipase needs to be evaluated, potentially by using approaches like the one presented herein. The enzyme activities of lipid kinases, phosphatases, and lipases are acutely modulated by hormones and growth factors. An important future direction is to identify physiological stimuli that use the PIP signaling pathway to regulate K_ATP channel activity.

Abbreviations

K_ATP channel

ATP-sensitive potassium channel

PI

phosphatidylinositol

PIP

phosphoinositide

PIP₂

PI 4,5-bisphosphate

PIP₃

PI-3,4,5-P₃

PIP5K

PI-4-phosphate 5-kinase

K_1/2

half-maximal inhibitory concentration

GFP

green fluorescent protein