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Originally published In Press as doi:10.1074/jbc.M403413200 on May 20, 2004

J. Biol. Chem., Vol. 279, Issue 36, 37271-37281, September 3, 2004
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Characteristic Interactions with Phosphatidylinositol 4,5-Bisphosphate Determine Regulation of Kir Channels by Diverse Modulators*

Xiaona Du{ddagger}, Hailin Zhang{ddagger}§, Coeli Lopes§¶¶, Tooraj Mirshahi§||**, Tibor Rohacs§||**, and Diomedes E. Logothetis§{ddagger}{ddagger}

From the {ddagger}Department of Pharmacology, Hebei Medical University, Shijiazhuang, China and the §Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York University, New York, New York 10029

Received for publication, March 27, 2004 , and in revised form, May 11, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The activity of specific inwardly rectifying potassium (Kir) channels is regulated by any of a number of different modulators, such as protein kinase C, Gq -coupled receptor stimulation, pH, intracellular Mg2+ or the {beta}{gamma}-subunits of G proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) is an essential factor for maintenance of the activity of all Kir channels. Here, we demonstrate that the strength of channel-PIP2 interactions determines the sensitivity of Kir channels to regulation by the various modulators. Furthermore, our results suggest that differences among Kir channels in their specific regulation by a given modulator may reflect differences in their apparent affinity of interactions with PIP2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Inwardly rectifying potassium (Kir)1 channels comprise a superfamily composed of seven subfamilies (Kir 1–7) containing at least 15 members in mammals. Kir channels are expressed in many tissues and serve important roles in cellular physiology such as cell excitability, K+ homeostasis, and insulin secretion. The activity of Kir channels is regulated by many intracellular factors and second messengers (1). Examples of the physiological importance of Kir regulation include: the ATP dependence of Kir6.X (KATP) channels in the control of insulin secretion (2, 3) and the determination of myocardial resistance to hypoxia (4, 5); the regulation of Kir3.X (GIRK) K+ channels by G-proteins that accounts for the vagal control of heart rate (e.g. Ref. 6); the regulation of Kir1.X (ROMK) channels by K+ and pH, which controls K+ secretion in the kidney (7), and the regulation of Kir4.1 channels in glial cells (8) and the inner ear (9), controlling K+ homeostasis and excitability.

A common feature of Kir channels that has emerged recently is that they all require the membrane phospholipid, PIP2 to maintain their activity (6, 10–13). PIP2, although only a minor component of the plasma membrane phospholipids, plays an important role in cell signaling; PIP2 serves as the substrate for PLC (phospholipase C)-mediated hydrolysis into the two ubiquitous second messengers diacylglycerol and inositol triphosphate. Recent studies have suggested that PIP2 directly mediates receptor-induced modulation of Kir channel activity (14, 15). Defects in channel-PIP2 interactions appear to underlie Andersen's and Bartter's syndrome (13).

The molecular mechanism by which Kir channels are regulated as a consequence of stimulation of different PLC isoforms, such as PLC{beta} coupled to receptor signaling through Gq- or PLC{gamma}-coupled to tyrosine kinase receptors, remains unsettled. Kir2.3, Kir3.X, and Kir 6.X channels have been shown to be inhibited through activation of PKC (16–20), directly by hydrolysis of PIP2 (14, 15, 21–23), or even directly by Mg2+ possibly acting as a second messenger (24). pH regulation of Kir channels has been extensively studied. It is generally believed that intracellular pH inhibits Kir channels through protonation of certain amino acid residues within the cytosolic channel domains (25–27). However, a mechanism other than mere protonation is needed to explain pH inhibition of Kir channels (26, 28). Recent studies indicate that PIP2 also plays an important role in the pH-induced inhibition of Kir channels (29–31).

In the present study, we aimed to explore the hypothesis that channel-PIP2 interactions determine modulation of Kir channel activity by multiple regulatory factors. We predominantly used two members of the Kir2 family, Kir2.1 (IRK1) and Kir2.3 (IRK3), and their mutants to show that the strength of channel-PIP2 interactions determines the degree of inhibition of these Kir2 channels by receptor (M1 and EGFR) stimulation, PKC activation (via PMA treatment), intracellular Mg2+ and pH. Our results strongly suggest that the apparent affinity of channel-PIP2 interactions is a key element in determining modulation of Kir channels.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular Biology—All cDNA constructs were subcloned into the pGEMHE plasmid vector and used as described. Point mutants were produced by Pfu mutagenesis with a QuikChange kit (Stratagene, La Jolla, CA). Sequences were confirmed by DNA sequencing. Recombinant Kir channels, M1 and EGF receptors were expressed in Xenopus laevis oocytes as previously described (35). cRNA was produced with T7 RNA polymerase using a kit (Ambion, Austin, TX). cRNA of the various Kir channels and their mutants and of receptors was injected in the range of 0.5–10 ng/oocyte depending on the functional expression level of the given construct.

Electrophysiology—Recordings in X. laevis oocytes were performed 2–4 days following cRNA injection.

Whole oocyte currents were measured by conventional two-microelectrode voltage clamp with a GeneClamp 500 amplifier (Axon Instruments). Electrodes were filled with 3 M KCl dissolved in 1% agarose to prevent the leakage of KCl into the oocytes. The electrodes had a resistance less than 1 M{Omega}. Oocytes were constantly perfused with a high potassium solution (ND96K) containing (in mM) 96 KCl, 1 NaCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES (pH 7.4). A low potassium solution (ND96) was used in some experiments to inhibit most of the Kir currents at –80 mV. ND96 contained (in mM) 96 NaCl, 1 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES (pH 7.4). Current amplitudes were measured at +80 mV and –80 mV. Data acquisition and analysis were carried out using pClamp8 (Axon Instruments) and Origin 6 (Microcal) software.

Macropatch channel activity was recorded from devitellinized oocytes under the inside-out mode of standard patch clamp methods using an EPC-9 or EPC-10 patch clamp amplifier and PULSE/PULSEFIT (version 7.6 or 8.5) data acquisition software (Heka Electronik, Germany). Electrodes were made from borosilicate glass (WPI) using a Sutter P-97 microelectrode puller and gave a tip diameter of 5–15 µm that had a resistance of 0.5–1M{Omega} when filled with an electrode solution containing (in mM) 96 KCl, 1 MgCl2, and 5 HEPES (pH 7.4). Three bath solutions were used: 1) an ATP-containing solution (in mM): 96 KCl, 5 EGTA, 1 MgATP and 10 HEPES (pH 7.4); 2) a FVPP solution (in mM): 96 KCl, 5 EDTA, 10 HEPES, 5 NaF, 3 Na3VO4, 10 Na2PO7 (pH 7.4) to prevent current rundown (10, 12); and 3) a ND96K solution (in mM): 96 KCl, 5 EGTA, and 10 HEPES (pH 7.4). Current amplitudes were measured at –80 mV with a sampling rate of 100 Hz. Data were analyzed using PULSEFIT and Origin software. Kinetics of inhibition by PIP2 antibody or polylysine have been previously justified as reliable measures of channel-PIP2 interactions and were used as previously described (13).

Chemicals—PIP2 was purchased from Roche Applied Science and Roche Diagnostics; DiC8 PIP2 was purchased from Echelon. PIP2 antibody was purchased from Assay Technologies (Ann Arbor, MI). PIP2 and PIP2 antibody were prepared as described previously (12, 13). All other chemicals were purchased from Sigma.

Error bars in the figures represent S.E. Each experiment shown or described was performed on 3–5 oocytes of the same batch. A minimum of 2–3 batches of oocytes was tested for each experiment shown. The unpaired t test was used to assess statistical significance in all cases where indicated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of M1 and EGF Receptors Inhibits Kir2.3 Channel but Not Kir2.1 Channel Currents—We chose the Kir2.1 and Kir2.3 channels as representatives to study Kir channel modulation. These two channels serve as good examples, because they have been shown to be modulated distinctly by M1 activation (24), PKC (32, 33), Mg2+ (24), and pH (28), even though they belong to the same subfamily of Kir channels.

When expressed in Xenopus oocytes, both Kir2.1 and Kir2.3 channels gave substantial inwardly rectifying currents. Fig. 1, A and B, shows the current traces recorded by two-electrode voltage clamp at +80 (above the dotted zero current line) and –80 mV (below the dotted zero current line) in a high extracellular K+ bath solution (ND96K). When M1 muscarinic receptors were coexpressed with these Kir channels and were stimulated by acetylcholine (ACh, 5 µM), Kir2.3 currents were consistently inhibited, whereas Kir2.1 currents were not (Fig. 1, A–C). Transient Ca2+-activated Cl– currents seen at +80 mV were indicative of PIP2 hydrolysis by PLC, which is known to be coupled to M1 receptor signaling (e.g. Ref. 14). Full recovery of Kir2.3 currents from inhibition could be obtained following long term washout of ACh (data not shown).



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  		FIG. 1.
ACh activation of M1 receptor inhibits Kir2.3 currents but not Kir2.1 currents and occludes EGF inhibition of Kir2.3 currents. Kir2.3 (A), Kir2.1 (B) channels, and M1 receptor were expressed in Xenopus oocytes. Currents at +80 mV (above dotted zero level) and –80 mV (below dotted zero level) were recorded using the two-electrode voltage clamp technique. High K solution (ND96K) was used in the bath. 5 µM ACh was applied to the bath for the period of time indicated by the bar. C shows the summary data of the channel inhibition measured at –80 mV. **, p < 0.01 compared with Kir2.3. D–F, M1 receptor and EGF receptor were coexpressed in oocytes. First application of EGF (100 ng/ml) (D) or ACh (E) followed by subsequent application of ACh or EGF, respectively. F shows summary data of Kir2.3 channel inhibition by application of both ACh and EGF measured at –80 mV.

 
We investigated whether PLC-mediated hydrolysis of PIP2 through a G protein-independent pathway would also result in Kir2.3 inhibition. In addition, we tested whether G protein-dependent and -independent pathways of PIP2 hydrolysis were occlusive of each other in inhibiting K+ currents. The epidermal growth factor receptor (EGFR) hydrolyzes PIP2 via direct coupling to PLC{gamma}. Kir2.3 channel, M1, and EGF receptors were coexpressed in oocytes and ACh (5 µM) and EGF (100 ng/ml) were applied through the bath solution. EGF induced a similar inhibition of Kir2.3 currents as ACh, and the inhibition of Kir2.3 by ACh or EGF was occlusive for the other agent (Fig. 1, D–F). EGF has been shown not to induce a significant inhibition of Kir2.1 currents (14). Thus, two independent pathways that hydrolyze PIP2 showed similar effects on Kir2.3 and Kir2.1 channels. Moreover, each pathway occluded the effect of the other in inhibiting Kir2.3 currents.

ACh- and EGF-induced Inhibition of Kir Currents Shows a Strong Correlation to the Apparent Affinity of Interaction with PIP2—We have previously identified mutations that alter channel-PIP2 interactions (e.g. Ref. 12 and 13). We first used PIP2 antibody to functionally assess channel-PIP2 interactions (10, 12). Fig. 2A shows the current traces recorded from inside-out macropatches held at –80 mV. PIP2 antibody inhibited Kir2.3 significantly faster than Kir2.1 currents, suggesting that Kir2.1 channels interact tighter with PIP2 than Kir2.3 channels do. We previously identified an amino acid difference between Kir3.4* and Kir2.1 channels, which is critical for interactions with PIP2 (12). Mutation of isoleucine in Kir3.4*(Ile229) to the corresponding Kir2.1 leucine (Leu222) strengthened interactions of Kir3.4* with PIP2. Kir2.3 channels also have an isoleucine (Ile213) in the position corresponding to GIRK4 (Ile229). We made the point mutation of Kir2.3 (I213L) and tested the kinetics of inhibition by PIP2 antibody. As shown in Fig. 2A, Kir2.3 (I213L) significantly slowed the time course of inhibition by PIP2 antibody. In addition, the point mutant Kir2.1(R312Q) was inhibited by PIP2 antibody with much faster kinetics than control (Fig. 2A), consistent with previous results (13). Summary data in Fig. 2B show the time needed for PIP2 antibody to inhibit half the channel currents.



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  		FIG. 2.
The apparent affinity of the channel-PIP2 interaction determines the degree of ACh- and EGF-induced inhibition of Kir channels. A, records from inside-out macropatches held at –80 mV; FVPP is used as the bath solution. PIP2 antibody inhibits Kir channels with different kinetics, indicating different strengths of interaction with PIP2. B, summary data for the experiment shown in A; the time needed for PIP2 antibody to inhibit Kir currents to half of their initial amplitudes. The large value indicates a strong interaction. C, sequence alignment of Kir channels. Kir2.3(I213) and homologous amino acids in other Kir channels have been highlighted. D, dose-response curves constructed from measurements of inside-out macropatches expressing the channels indicated responding to applications of different concentrations of the diC8 PIP2. Solid lines are Hill fits to the data points for the particular channel. Each data point is an average of at least three experiments. Data are expressed as a percentage of the maximal response as determined by the Hill fit. E, summary of M1-mediated inhibition from whole cell recordings using two-electrode voltage clamp technique. **, p < 0.01 compared with Kir2.3; ##, p < 0.01 compared with Kir2.1. F, summary of EGF-mediated inhibition from whole cell recordings using the two-electrode voltage clamp technique. **, p < 0.01 compared with Kir2.3; ##, p < 0.01 compared with Kir2.1.

 
Thus, Kir2.3(I213L) behaved similarly to Kir3.4(I229L) in showing slower kinetics of PIP2 antibody inhibition of these mutant currents compared with control. In fact, there is good correlation of the residue type at this position (Fig. 2C, highlighted) with the strength of interaction of PIP2 with Kir channels: channels possessing a Leu residue generally show stronger interactions with PIP2 than channels possessing an Ile residue at this position (see Fig. 4C and "Discussion"). We proceeded to test whether this mutation caused an increase in the apparent affinity of Kir2.3 to PIP2. We used the 8-carbon long acyl chains on PIP2 (DiC8), a synthetic water soluble analog of PIP2, to construct dose-response curves of PIP2 on channel activity. Shifts in the PIP2 dose-response curves for Kir currents with specific mutations affecting channel-PIP2 interactions have previously suggested changes in the channel-PIP2 apparent affinity (13). Consistent with the PIP2 antibody results (i.e. strength of interactions of Kir2.1 > Kir2.3(I213L) > Kir2.3) (Fig. 2D), the relative apparent affinities of these channels for PIP2 were in the same order (Kir2.1:EC50 ~5 µM > Kir2.3(I213L):EC50 ~8 µM > Kir2.3:EC50 ~29 µM). Given these results we will use interchangeably the terms "strength" of channel-PIP2 interactions or "apparent affinity" of a channel to PIP2.



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  		FIG. 4.
ACh- and PMA-induced inhibition of Kir currents. A, percent ACh-induced current inhibition from two-electrode voltage clamp recordings from members of all Kir subfamilies expressed in Xenopus oocytes. B, percent PMA-induced current inhibition from two-electrode voltage clamp recordings from members of all Kir subfamilies expressed in Xenopus oocytes. C, relative strength of channel-PIP2 interactions for Kir channels. Summary data (n = 3–9) of T50 values for polylysine inhibition for each of the channels tested.

 
We next proceeded to test whether these mutations that altered channel-PIP2 interaction also affected modulation of Kir2.3 and Kir 2.1 currents by M1 and EGFR stimulation. Fig. 2, E and F, shows that Kir2.3(I213L) was inhibited significantly less than Kir2.3 by either M1 or EGFR stimulation, whereas Kir2.1(R312Q) showed greater inhibition than Kir2.1 by M1 and EGFR stimulation. Therefore, we conclude that the extent of agonist-induced current inhibition of these Kir channels is controlled by the apparent affinity of interactions between the channels and PIP2.

PMA- and ACh-induced Inhibitions of Kir Currents Show a Strong Correlation with the Apparent Affinity of a Channel to PIP2—Previous studies have shown that Kir2.3 channels are inhibited by PMA through PKC-mediated phosphorylation (32, 33). In contrast, Kir2.1 channels are generally not modulated by PKC activation. We performed similar experiments to those described above using PMA-modulated Kir channels expressed in oocytes. When PMA (100 nM) was applied to oocytes expressing Kir2.3 channels, currents were inhibited irreversibly; the inhibition started with a delay of ~2 min and reached a plateau 4–5 min following PMA application (Fig. 3A); Kir2.3 currents were inhibited to about 50% of their original amplitude (Fig. 3E) and no further inhibition was observed after PMA concentration was increased from 100 nM to 1 µM; the inactive form of PMA, 4-{alpha}-PMA, had no effect on Kir2.3 currents (data not shown); a specific PKC blocker, bisindoylmaleimide, prevented PMA-induced inhibition of Kir2.3 currents (Fig. 3E). These results suggest that PMA inhibited Kir2.3 currents through a PKC-mediated mechanism. In contrast, PMA did not inhibit significantly Kir2.1 currents (Fig. 3, B and E). Kir2.3(I213L), which increased the apparent affinity of interactions with PIP2, completely abolished PMA-induced inhibition (Fig. 3, C and E). In contrast, Kir2.1(R312Q), which showed decreased affinity of interactions with PIP2, did exhibit inhibition by PMA (Fig. 3, D and E). Thus, apparent affinity of channel-PIP2 interactions determines modulation of these Kir channels by PMA.



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  		FIG. 3.
The apparent affinity of the channel to PIP2 determines the degree of PMA-induced inhibition of Kir channels. Whole cell currents recorded using the two-electrode voltage clamp technique. Current traces at –80 (below dotted line) and +80 mV (above the dotted line) are shown. Representative current traces recorded from oocytes expressing (A) Kir2.3, (B) Kir2.1, (C) Kir2.3(I213L), and (D) Kir2.1(R312L) channels are shown. PMA (100 nM) was applied through the bath solution as indicated. E, summary data for PMA-induced inhibition. **, p < 0.01 compared with Kir2.3; ##, p < 0.01 compared with Kir2.1. F, basal Kir3.4* inhibited by ACh (left) or PMA (right) application in a two-electrode voltage clamp technique experiment. G, co-expression of G{beta}{gamma} reduces greatly inhibition of basal Kir3.4* currents by ACh (left) or PMA (right). H, summary data for the reduction of ACh (left) and PMA-induced (right) inhibition with G{beta}{gamma} coexpression **, p < 0.01 compared with channels expressed alone.

 
To further examine the generality of our conclusion, we tested for ACh- and PMA-induced inhibition in the presence and absence of G{beta}{gamma}. Activation of Kir3.4* channels by G{beta}{gamma} (10, 12) or intracellular Na+ (6, 12, 34) constitutes a physiological way by which channel-PIP2 interactions can be strengthened. Fig. 3 also shows that basal Kir3.4 currents could be inhibited by either ACh or PMA treatment (Fig. 3F). Coexpression of Kir3.4* channels with G{beta}{gamma} greatly reduced the ACh- or PMA-induced current inhibition (Fig. 3G). These results further support the conclusion that the strength of channel-PIP2 interactions determines modulation of Kir channels by ACh and PMA. We next examined the ACh-(Fig. 4A) and PMA-induced (Fig. 4B) inhibition of most Kir channel family members. We correlated these effects to the strength of interaction with PIP2 exhibited by each of these channels (Fig. 4C), as assessed by polylysine block kinetics (see Ref. 13). So far, three Kir channels have not yielded homomeric currents when expressed in Xenopus oocytes (e.g. Kir3.1, Kir5.1 and Kir6.1, or Kir6.1{Delta} 36). Mutants of Kir3 family members have been shown to exhibit large homomeric currents (Kir3.1-F137S and Kir3.4-S143T) (35, 36). Heteromeric expression often also enhances channel targeting to the cell surface and yields larger currents than the corresponding homomeric ones (e.g. Kir3.1/3.4 and Kir4.1/5.1). We studied 11 homomeric and two heteromeric channels (Fig. 4). Based on our results, we ranked strength of channel-PIP2 interactions relative to the strongest interacting channel Kir4.1. This analysis indicated the following rank order among Kir channel subfamilies (from the strongest to the weakest PIP2 interacting): Kir4 > Kir1 ~ Kir2 > Kir6 > Kir7 ~ Kir3 (Fig. 4C). Both the ACh- and PMA-induced effects showed an almost perfect inverse correlation to the strength of channel-PIP2 interactions (Fig. 4, A and B). The only exception seemed to be Kir2.2, which displayed relatively strong channel-PIP2 interactions with disproportionately large ACh and PMA current inhibition. These results suggest that the strength of channel-PIP2 interactions determines modulation of Kir channels through agonist- and PMA-induced effects.

Enhanced Channel-PIP2 Interaction and PIP2 Prevent Intracellular Mg2+ Inhibition of Kir2.3 Channels—Intracellular Mg2+ was shown previously to inhibit Kir2.3 channel currents, and was suggested to act as a second messenger accounting directly for the M1-induced inhibition of Kir2.3 channels (24). In the present study we tested whether the Mg2+-induced inhibition of Kir2.3 currents was PIP2-dependent. Data shown in Fig. 5 were obtained from inside-out macropatches. Kir2.3 as well as Kir2.1 channels generally gave currents in the nanoampere range when recorded in the cell-attached mode at –80 mV (e.g. Fig. 5A). When an inside-out patch was excised into a Mg2+-free high K+ solution (ND96K), Kir2.3 currents ran up first, reaching a plateau that was maintained for a short time before starting to run down. Kir2.3 currents were more stable in a FVPP solution, which inhibits phosphatases and thus prevents breakdown of PIP2 (10). Under such conditions, 1 mM Mg2+ significantly accelerated rundown of Kir2.3 currents when applied to the inner surface of inside-out patches. Only a small portion of the rundown currents could be recovered after Mg2+ was washed away (Fig. 5A). However, the time course of Kir2.3 current rundown was variable from patch to patch, possibly reflecting variable phosphatase activities. PIP2 (10 µM), applied to the inner surface of patches, could activate Kir2.3 currents after they completely ran down; normally long PIP2 applications were required to reactivate Kir2.3 channels, possibly because of the slow diffusion of exogenous PIP2 into the membranes and binding to the channels. Once Kir2.3 channels were reactivated by PIP2, Mg2+ could not induce current rundown any more. Instead, Mg2+ application following reactivation by PIP2 induced a nonspecific reversible inhibition. Kir2.3(I213L) currents were less sensitive to Mg2+ inhibition. Fig. 5B shows that each application of Mg2+ accelerated current rundown but the Mg2+-induced rundown was much slower for Kir2.3(I213L) than the Kir2.3 control. After rundown, PIP2 activated Kir2.3(I213L) faster than Kir2.3 currents, suggesting a stronger binding affinity of Kir2.3(I213L) channels to PIP2; Mg2+ again could not significantly inhibit Kir2.3(I213L) currents after they were reactivated by PIP2. In previous work (24), Kir2.1 channels were assessed to be Mg2+-insensitive. In our experiments, we found Mg2+ able to induce Kir2.1 current rundown, albeit with much slower kinetics (Fig. 5C). PIP2 reactivated Kir2.1 currents following run-down with fast kinetics, and Mg2+ induced a small, reversible inhibition of the currents that was comparable with the inhibition of Kir2.3 or Kir2.3(I213L) currents following PIP2 reactivation.



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  		FIG. 5.
The strength of channel-PIP2 interactions and presence of PIP2 determine Mg2+ inhibition of Kir channels. Inside-out macropatches held at –80 mV. Mg2+ (1 mM) and PIP2 (10 µM) were applied via the bath as indicated. The dotted lines indicate zero current level. Arrows indicate the time when an inside-out patch was excised. Mg2+ inhibition of Kir2.3 (A), Kir2.3(I213L) (B), Kir2.1 (C), and Kir2.1(R312Q) (D) currents was measured at the end of the 1-min application, and was summarized in E. **, p < 0.01 compared with Kir2.3; ##, p < 0.01 compared with Kir2.1.

 
Fig. 5D shows that the Mg2+-induced rundown was much faster for Kir2.1(R312Q) than for Kir2.1 wild type. After run-down, PIP2 reactivated Kir2.1(R312Q) slower than Kir2.1 currents, suggesting a weaker binding affinity for Kir2.1(R312Q) than for Kir2.1. Once again, Mg2+ induced only a PIP2-independent inhibition following reactivation by PIP2.

Fig. 5E shows summary data of the inhibition of Kir2.3, Kir2.3(I213L), Kir2.1, and Kir2.1(R312Q) by Mg2+ before and after application of PIP2. The data are quantified 1 min following application of Mg2+.

We next proceeded to use an ATP-containing solution to test if ATP, in the presence of which endogenous synthesis of PIP2 is possible, would reduce Mg2+-induced inhibition of the Kir currents in a manner similar to that obtained following PIP2 application. Inside-out macropatches expressing Kir2.3, Kir2.3(I213L), Kir2.1, and Kir2.1(R312Q) channels were excised into 1 mM MgATP solution and 1 mM Mg2+ was applied to the patches for a short time (1 min, Fig. 6, A–D). In the presence of MgATP, Mg2+ had a small inhibitory effect on Kir2.3(I213L), Kir2.1, and Kir2.1(R312Q) currents (Fig. 6, B–D), similar to the inhibition for these channels shown in Fig. 5, B–D, that occurred in the presence of PIP2. Kir2.3(I213L), Kir2.1, and Kir2.1(R312Q) currents were stable in the MgATP solution. However, Kir2.3 currents ran down even in the presence of MgATP and thus Mg2+ inhibited Kir2.3 (Fig. 6A) currents more than the corresponding Kir2.3(I213L). The Mg2+-induced inhibition was less in MgATP-containing (Fig. 6A) than in MgATP-free (Fig. 5A) solution. Summary data in Fig. 6E show the percentage inhibition of the Kir channels by Mg2+ measured at the end of the first application of 1 mM Mg2+. The data shown in Figs. 5 and 6 strongly suggest that the strength of channel-PIP2 interactions and the level of membrane PIP2 determine Mg2+ modulation of the Kir channels.



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  		FIG. 6.
MgATP reduces Mg2+-induced inhibition of Kir channels. A–D, inside-out macropatches held at –80 mV. Inside-out patches were excised into MgATP solution. Arrows indicate the time when an inside-out patch was excised. The dotted lines indicate the zero current level. Summarized data in E were measured at the end of a 1-min application of Mg2+. **, p < 0.01 compared with Kir2.3; #, p > 0.05 compared with Kir2.1.

 
Enhanced Channel-PIP2 Interactions and Presence of PIP2 Prevent Intracellular Proton Inhibition of Kir Channels— Kir2.3 channels have been shown to be regulated by intracellular pH, and multiple histidines in the C terminus of the channel have been suggested to serve as the proton action sites (28). We investigated whether pH regulation of Kir2.3 channels is PIP2-dependent. Both whole cell and inside-out macropatch recordings were used in the experiments. In whole cell recordings, we used azide (sodium salt) and KHCO3 to lower intracellular pH. Azide is a metabolic inhibitor and has been used to activate KATP channels expressed in Xenopus oocytes by lowering internal ATP concentration (37). When oocytes expressing Kir2.3 channels were treated with 3 mM azide, a fast and reversible inhibition of Kir2.3 currents was seen (Fig. 7A). We considered this inhibition to be the result of lowered intracellular pH, because: 1) metabolic inhibitors are known to decrease intracellular pH (38); 2) injection of 20 mM HEPES into the oocytes largely prevented the inhibition (data not shown); 3) Kir2.3 currents were directly inhibited by intracellular acidification (see below); and 4) Kir3.4 channels that were activated by intracellular acidification were also activated by azide.2 Recently, it was reported that metabolic inhibitors including azide inhibited Kir2 (2.2 and 2.3) channels through a pH-mediated process (39). We used azide mainly because of its pathophysiological relevance to the conditions like ischemia and hypoxia. KHCO3 has also been used previously as a modulator of intracellular pH (25, 40). As it can be seen in Fig. 7A, when KCl in the bath solution was replaced by KHCO3 (pH in the solution was adjusted to 7.4), a larger inhibition for Kir2.3 currents was seen than for Kir2.3(I213L); the inhibition was fast and reversible. A low-potassium solution (ND96) was applied at the end of the recording to set the baseline for current measurements. Whereas KHCO3 induced smaller inhibition of Kir2.3(I213L) currents, azide did not inhibit Kir2.3(I213L) currents at all (Fig. 7B). Azide failed to inhibit Kir2.1 currents or Kir2.1(R312Q) currents; the intracellular pH change induced by azide may be too small to distinguish the differential sensitivity of Kir2.1 and Kir2.1(R312Q) channels to pH (see Fig. 8E, pH 6.8); KHCO3 induced a larger inhibition on Kir2.3(I213L) currents than on Kir2.1 currents (Fig. 7, C and D). Summary data are shown in Fig. 7E.



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  		FIG. 7.
The strength of channel-PIP2 interactions determines azide- and KHCO3-induced inhibition of Kir channels. Whole cell currents recorded using the two-electrode voltage clamp technique. Current traces at –80 (below dotted line) and +80 mV (above dotted line) are shown. Azide (3 mM) and KHCO3 (96 mM) were applied through the bath solution as indicated. Azide and KHCO3 inhibit Kir2.3 (A), Kir2.3(I213L) (B), Kir2.1 (C), and Kir2.1(R312Q) (D) currents in correlation with their strength of interaction with PIP2. E, summary data showing normalized remaining currents after azide- and KHCO3 -induced inhibition. The dotted line showsthe current level before azide and KHCO3 application. **, p < 0.01 compared with Kir2.3.

 



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  		FIG. 8.
Intracellular pH inhibits Kir currents in a manner dependent on the strength of channel-PIP2 interactions and the presence of PIP2. Inside-out macropatches held at –80 mV. FVPP or MgATP solution is the bath solution. The bath solution was changed to different pH values as indicated. Acidic pH inhibits Kir2.3 (A), Kir2.3(I213L) (B), Kir2.1 (C), and Kir2.1(R312Q) currents in correlation with their strength of interaction with PIP2. The dotted line indicates zero current level. E, the current-pH relationship was fitted by a Hill function, I = 1/(1 + (pK/pH)n, where pK is the pH at which the current is 50% inhibited and n is the Hill coefficient. F, pH 6.5 inhibition of Kir2.3 currents followed activation by PIP2. pH 6.5 solution was applied multiple times as indicated. The dotted line indicates zero current level.

 
We next tested pH sensitivity of Kir2.3, Kir2.3(I213L), Kir2.1, and Kir2.1(R312Q) channel currents in inside-out macropatches. Fig. 8 shows that Kir currents were inhibited by decreasing pH with varying sensitivities, which again correlated well with their strength of interaction with PIP2. Fig. 8E shows dose-response relationship curves between Kir channel currents and intracellular pH, which were fitted by a Hill equation. Kir2.3, Kir2.3(I213L), Kir2.1, and Kir2.1(R312Q) were inhibited by decreasing the pH with pK (i.e. the pH value for half-maximal current inhibition) of 6.77, 6.27, 4.93, and 5.72, respectively. Hill coefficients were 2.4, 1.9, 1.9, and 2.2 for Kir2.3, Kir2.3(I213L), Kir2.1, and Kir2.1(R312Q) currents, respectively. We then tested if Kir 2.3 currents would become less sensitive to proton inhibition following activation by PIP2. Fig. 8F shows such an experiment. In this recording, Kir2.3 currents were inhibited when the bath pH was changed briefly from 7.4 to 6.5; the proportion of the current inhibited at pH 6.5 was increased during current rundown (e.g. the second application of pH 6.5 solution reduced currents to a greater extent than the first application), indicating a dependence on membrane PIP2 that was presumably lost gradually after patch excision. PIP2 reactivated the currents that became less and less sensitive to the pH 6.5-induced inhibition during the slow reactivation course. In four patches, pH 6.5 inhibited 22 ± 7.4% of Kir2.3 currents in the presence of PIP2, which is significantly (p < 0.01) less than 79 ± 11% inhibition of the currents in the absence of PIP2. These results strongly suggested that internal pH regulated the Kir channels in a PIP2-dependent manner.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we show that the strength of channel-PIP2 interactions controls the sensitivity of Kir channels to regulation by receptor stimulation, PKC, intracellular Mg2+, and pH. Our results suggest that differences in the regulation of Kir channels (e.g. phosphorylation, Mg2+, and pH binding sites) may reflect differences in the strength of channel-PIP2 interactions.

Kir Channel-PIP2 Interaction and Kir Channel Modulation by Receptor or PMA Activation—Our data clearly show that the affinity of channel-PIP2 interactions controls inhibition of Kir2.1 and Kir2.3 channels by receptor stimulation and PMA. We found that Kir2.3 channels were otherwise inhibited by agonist and PMA stimulation, and became insensitive to these modulators when the affinity of their interaction with PIP2 was increased by the single point mutation Kir2.3 (I213L); on the other hand, Kir2.1 channels, which are normally not inhibited by agonist and PMA, became sensitive to inhibition when the affinity of their interaction with PIP2 was decreased by the single point mutation Kir2.1(R312Q). Kir2.3(I213L) is homologous to Kir3.4(I229L), a mutant we identified in previous work and showed that it enhanced the affinity of Kir3.4 channel interactions with PIP2 (12). Fig. 2C compared the homologous amino acid residue of Kir2.3(I213) to all other Kir channels (highlighted). Kir channels (Kir2.2, Kir2.3, Kir3 family, and Kir6 family) that have an isoleucine in this position generally have weak or intermediate affinity interactions with PIP2 (10, 12; Fig. 4C), and are modulated by receptor or PKC activation (18, 20, 24, 41–43) (Fig. 4, A and B); Kir channels (Kir1.1, Kir2.1, Kir 2.4, Kir4.1, and Kir4.2) that have a leucine in this position generally show high affinity interactions with PIP2 (10, 12) (Fig. 4C), and are not modulated by agonist or PKC activation (24) (Fig. 4, A and B). It is likely that an isoleucine or leucine at this position plays an important role in affecting Kir channel interactions with PIP2 in all these Kir channels. One exception to this correlation seems to be Kir7.1 channel, which although has a leucine at this position shows weak affinity interactions with PIP2 (Fig. 4C). Accumulating evidence supports the hypothesis that channel-PIP2 interaction are electrostatic in nature, where the negatively charged phosphate groups of the phospholipid interact with positively charged amino acids in the Kir protein (12, 13, 44, 45). The effects of isoleucine or leucine residues ought to be indirect, as these residues are not charged. It is likely that these residues exert their effects by influencing the way neighboring residues interact with PIP2. Five positions upstream of the isoleucine or leucine residues there is a positively charged arginine (at the beginning of the sequence shown in Fig. 2B) that is conserved in all Kir channels. Our previous work demonstrated that this arginine is a crucial residue in the interactions of Kir channels with PIP2 (12, 13). When we map these two residues (arginine and isoleucine or leucine) to the structure of a recently crystallized C terminus of the Kir3.1 channel (46), they are co-localized to positions close to the inner membrane (not shown). A leucine could be stabilizing the interaction of the arginine with PIP2, whereas an isoleucine could be destabilizing this interaction. Arginine 312 in Kir2.1 is another important PIP2-interacting residue previously identified (13). We chose to mutate this residue because even though Kir2.1(R312Q) had a weaker interaction with PIP2, it still gave measurable currents to study, whereas mutation of other PIP2-interacting residues (e.g. R218Q) often resulted in channels with currents too small to be reliably measured in most batches of oocytes.

The mechanism underlying inhibition of Kir channels by receptor (Gq-coupled or growth factor) stimulation is still under debate. Cell signaling through receptor activation involves PIP2 hydrolysis and subsequent activation of its downstream product PKC. Direct PIP2 hydrolysis has been implicated in Kir channel inhibition (14, 15, 21–23). However, a PKC-mediated mechanism has also been suggested (16–19), because receptor-mediated inhibition can be prevented by PKC inhibitors and mimicked by PMA that is known to activate PKC. A few attempts have been made to identify potential phosphorylation sites of PKC on Kir channels. Whereas most studies have failed to identify any PKC phosphorylation sites (24, 32, 42), one study has reported that a single residue, threonine at the N terminus of Kir2.3 (Thr53) is responsible for PMA-mediated inhibition; mutation of Kir 2.3(Thr53) to the corresponding residue of Kir2.1 (Kir 2.3(T53I) completely eliminated the response of the channel to PMA; the reverse mutation conferred Kir2.1(I79T) with sensitivity to PMA almost identical to Kir2.3. This threonine is not a known PKC phosphorylation site and it was not clear how it affected the response of the channel to PMA (33). One possibility is that Thr53 may behave like Ile213 that we identified in this study, namely to affect channel-PIP2 interactions. PIP2-interacting sites have been identified in the N terminus of Kir channels (13) where Thr53 resides. In light of all the findings so far, it seems unlikely that specific PKC phosphorylation site(s) exist only in channels sensitive to PKC modulation; it is more likely that common PKC site(s) exist in most Kir channels, and only channels that have a weaker interaction with PIP2 would allow PKC-mediated phosphorylation to take place or to be manifested. The assumption here is that the PMA effect seen on Kir channels is indeed through PKC activation (see Ref. 47), and PKC acts on the channel directly, not through some accessory molecules (48). As for the mechanism of receptor-mediated inhibition of Kir channels, our data suggest the following scenario: receptor activation could first hydrolyze PIP2 reducing the apparent affinity of the channel to PIP2; however, for those channels that have a high apparent affinity to PIP2, the remaining interaction is still strong enough to prevent PKC-mediated inhibition; for those channels that have a lower apparent affinity to PIP2, the remaining interactions are weakened to a point that PKC action is permitted.

Mg2+ Inhibition of Kir Currents Depends on PIP2—It was suggested that receptor (M1)-induced inhibition of Kir2.3 channels proceeded through intracellular Mg2+ acting as a direct second messenger (24). This conclusion was based mainly on the fact that M1 inhibited Kir2.3 currents but not Kir2.1 currents, an effect that could be mimicked by intracellular Mg2+. Partial exchange of the N and C termini of Kir2.1 channels with those from Kir2.3 conferred the chimeric channel with sensitivity to M1 and Mg2+ inhibition. These results are consistent with our present data. However, we argue that the different sensitivity of Kir2.1 and Kir2.3 channels to intracellular Mg2+ is a reflection of their different interaction properties with PIP2. In our experiments, Mg2+ inhibition of Kir channels exhibited two distinct characteristics. First, in the absence of PIP2, Mg2+ normally inhibited Kir channels irreversibly. Second, the only difference between Kir2.1 and Kir2.3 channels was that Mg2+ took a longer time to inhibit Kir2.1 currents (Fig. 5, A and C). Increasing the strength of PIP2 interactions, as with Kir2.3(I213L), slowed the Mg2+-induced inhibition of K+ currents, whereas decreasing the strength of these interactions, as with Kir2.1(R312Q), accelerated the kinetics of block (Fig. 5, A–E). In the presence of PIP2, Mg2+ inhibited mutant and wild-type Kir2.1 and Kir2.3 currents reversibly and with a similar potency (Fig. 5, A–D). These data are consistent with the notion that Mg2+, a known phosphatase activator, induced Kir channel inhibition through enhancing PIP2 hydrolysis and this effect could be compensated by exogenously applied PIP2; Mg2+, also a known Kir channel pore blocker (e.g. Ref. 49), rapidly and reversibly blocked Kir channels in the presence of PIP2. We also attempted to use a MgATP-containing solution to mimic the physiological conditions, where PIP2 would be synthesized endogenously. However, MgATP was apparently not sufficient to prevent Kir2.3 currents from continuously running down (Fig. 6A), which made assessment of the Mg2+ action difficult. Nevertheless, it is apparent in the records that Mg2+ induced a smaller inhibition of Kir currents in the presence of MgATP (Fig. 5). Although the possibility that Mg2+ plays a direct role in the M1-induced inhibition of Kir2.3 channels cannot completely be excluded, it is likely that the predominant effect of Mg2+ is mediated by enhancing hydrolysis of PIP2.

pH Regulation of Kir Channels Depends on PIP2—Our data indicate that intracellular acidic pH inhibits Kir channels in a PIP2-dependent manner. In both whole cell and inside-out patch recordings, inhibition of Kir2.3, Kir2.3(I213L) Kir2.1, and Kir2.1(R312Q) currents correlated well with the strength of channel-PIP2 interactions. Furthermore, in the presence of PIP2, Kir2.3 currents became less sensitive to pH inhibition (Fig. 8E). A number of Kir channels have been shown to be regulated by intracellular pH; the Kir channels that are inhibited by intracellular pH include Kir1.1 channels (25, 26, 31), Kir2.3 channels (28, 39, 40), Kir4.1 and Kir5.1 channels (30); Kir 6.1 and Kir6.2 channels, on the other hand, are activated by intracellular proton in a certain concentration range (50). Kir1.1 is the best studied channel with regard to understanding the mechanism of pH inhibition. A lysine (Lys80) was identified in the N terminus of the Kir1.1 channel that serves as a pH sensor (25). However, a lysine residue has a nominal pKa of 10.5, whereas Kir1.1 is inhibited by intracellular pH with a pKa of 6.8. An "Arg-Lys-Arg triad" hypothesis has been proposed to explain this three-order of magnitude discrepancy (26): two arginines in the N (Arg41) and C (Arg311) terminus of Kir1.1 channels form the "triad" with Lys80 that shifts the pKa of the lysine into the physiological range via electrostatic interactions. It is interesting to note that Arg311 in Kir1.1 channels is homologous to Arg312 in Kir2.1, a PIP2 interaction site (13) (Fig. 3A). More recent studies suggest that PIP2 plays an important role in the pH inhibition of Kir1.1 channels (29, 31); it was reported that the absence of a N-terminal PIP2 interaction site was a prerequisite for pH inhibition of Kir1.1 channels in the physiological range; introduction of an arginine into this site increased affinity of Kir1.1 channels to PIP2 and shifted the pKa of pH inhibition of Kir1.1 channels to a much lower value (31). A PIP2 dependence was also observed for pH inhibition of Kir4.1-Kir5.1 channel currents (30). Thus, our data combined with the above reports suggest that channel-PIP2 interactions may act like a switch that controls pH inhibition of Kir channels.

The current study presents a general mechanism underlying Kir channel regulation. PIP2, a membrane phospholipid that is known to be important in maintaining Kir channel activity, may act as the final checkpoint for regulation of Kir channels by other modulators, which may have action sites on Kir channels common to all or specific to one Kir channel. On the other hand, although it is less likely, the Kir channel modulators we discussed here may also influence Kir channel-PIP2 interactions and thus regulate Kir channel activities. Two recently published papers show that PKC activation inhibits Kir channel activity in association with a reduction in the membrane PIP2 level (51, 52), through a yet unknown mechanism. In either case, channel-PIP2 interactions play a critical role in the modulation of Kir channel activity.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant HL59949 (to D. E. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

¶¶ Supported by a grant-in-aid from the American Heart Association. Back

|| Supported by NRSA postdoctoral fellowships from the National Institutes of Health. Back

** Supported by an American Heart Association Scientist Development Grant. Back

Supported by National Science Foundation of China Grant 30270361 and Hebei Nature Science Foundation Grant 303464. To whom correspondence may be addressed. Tel.: 86-311-6265562; Fax: 86-311-6057291; E-mail: zhanghl{at}hebmu.edu.cn. {ddagger}{ddagger} Established Investigator of the American Heart Association. To whom correspondence may be addressed. Tel.: 212-241-6284; Fax: 212-860-3369; E-mail: diomedes.logothetis{at}mssm.edu.

1 The abbreviations used are: Kir, inwardly rectifying K+; ACh, acetylcholine; EGF, epidermal growth factor; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; EGFR, epidermal growth factor receptor. Back

2 X. Du, H. Zhang, and D. E. Logothetis, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Xixin Yan for oocyte isolation and members of the Logothetis laboratory for reading the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ruppersberg, J. P. (2000) Pflugers Arch. 441, 1–11[CrossRef][Medline] [Order article via Infotrieve]
  2. Ashcroft, F. M., Harrison, D., and Ashcroft, S. J. (1984) Nature 312, 446–448[CrossRef][Medline] [Order article via Infotrieve]
  3. Ashcroft, F. M., and Rorsman, P. (1989) Prog. Biophys. Mol. Biol. 54, 87–143[CrossRef][Medline] [Order article via Infotrieve]
  4. Benndorf, K., Schwalb, M., and Hirche, H. (1990) Pflügers Arch. 416, 207–209[CrossRef][Medline] [Order article via Infotrieve]
  5. Brown, A. M. (1997) Trends Cardiovasc. Med. 7, 118–124[CrossRef]
  6. Sui, J. L., Petit-Jacques J., and Logothetis, D. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1307–1312[Abstract/Free Full Text]
  7. Wang, W., Hebert, S. C., and Giebisch, G. (1997) Annu. Rev. Physiol. 59, 413–436[CrossRef][Medline] [Order article via Infotrieve]
  8. Ishii, M., Horio, Y., Tada, Y., Hibino, H., Inanobe, A., Ito, M., Yamada M., Gotow, T., Uchiyama, Y., and Kurachi, Y. (1997) J. Neurosci. 17, 7725–7735[Abstract/Free Full Text]
  9. Hibino, H., Horio, Y., Inanobe, A., Doi, K., Ito, M., Yamada, M., Gotow, T., Uchiyama, Y., Kawamura, M., Kubo, T., and Kurachi, Y. (1997) J. Neurosci. 17, 4711–4721[Abstract/Free Full Text]
  10. Huang, C. L., Feng, S., and Hilgemann, D. W. (1998) Nature 391, 803–806[CrossRef][Medline] [Order article via Infotrieve]
  11. Shyng, S. L., and Nichols, C. G. (1998) Science 282, 1138–1141[Abstract/Free Full Text]
  12. Zhang, H., He, C., Yan, X., Mirshahi, T., and Logothetis, D. E. (1999) Nat. Cell Biol. 1, 183–188[CrossRef][Medline] [Order article via Infotrieve]
  13. Lopes, C. M., Zhang, H., Rohacs, T., Jin, T., Yang, J., and Logothetis D. E. (2002) Neuron 34, 933–944[CrossRef][Medline] [Order article via Infotrieve]
  14. Kobrinsky, E., Mirshahi, T., Zhang, H., Jin, T., and Logothetis, D. E. (2000) Nat. Cell Biol. 2, 507–514[CrossRef][Medline] [Order article via Infotrieve]
  15. Xie, L. H., Horie, M., and Takano, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 15292–15297[Abstract/Free Full Text]
  16. Sharon, D., Vorobiov, D., and Dascal, N. (1997) J. Gen. Physiol. 109, 477–490[Abstract/Free Full Text]
  17. Stevens, E. B., Shah, B. S., Pinnock, R. D., and Lee, K. (1999) Mol. Pharamcol. 55, 1020–1027[Abstract/Free Full Text]
  18. Leaney, J. L., Dekker, L. V., and Tinker, A. (2001) J. Physiol. 534, 367–379[Abstract/Free Full Text]
  19. Perillan, P. R., Chen, M., Potts, E. A., and Simard, J. M. (2002) J. Biol. Chem. 277, 1974–1980[Abstract/Free Full Text]
  20. Thorneloe, K. S., Maruyama, Y., Malcolm, A. T., Light, P. E., Walsh, M. P., and Cole, W. C. (2002) J. Physiology 541, 65–80[Abstract/Free Full Text]
  21. Meyer, T., Wellner-Kienitz, M. C., Biewald, A., Bender, K., Eickel, A., and Pott, L. (2001) J. Biol. Chem. 276, 5650–5658[Abstract/Free Full Text]
  22. Haruna, T., Yoshida, H., Nakamura, T. Y., Xie, L. H., Otani, H., Ninomiya, T., Takano, M., Coetzee, W. A., and Horie, M. (2002) Circ. Res. 91, 232–239[Abstract/Free Full Text]
  23. Okitani, N., Kakei, M., Ichinari, K., Okamura, M., Miyamura, A., Nakazaki, M., Ito, S., and Tei, C. (2002) Am. J. Physiol. 282, H757–H765
  24. Chuang, H. H., Jan, Y. N., and Jan, L. Y. (1997) Cell 89, 1121–1132[CrossRef][Medline] [Order article via Infotrieve]
  25. Fakler, B., Schultz, J., Yang, J., Schulte, U., Brändle, U., Zenner, H. P., Jan, L. Y., and Ruppersberg, J. P. (1996) EMBO J. 15, 4093–4099[Medline] [Order article via Infotrieve]
  26. Schulte, U., Hahn, H., Konrad, M., Jeck, N., Derst, C., Wild, K., Weidemann, S., Ruppersberg, J. P., Fakler, B., and Ludwig, J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 15298–15303[Abstract/Free Full Text]
  27. Chanchevalap, S., Yang, Z. J., Cui, N. R., Qu, Z. Q., Zhu, G. Y., Liu, C. X., Giwa, L. R., Abdulkadir, L., and Jiang, C. (2000) J. Biol. Chem. 275, 7811–7817[Abstract/Free Full Text]
  28. Qu, Z., Yang, Z., Cui, N., Zhu, G., Liu, C., Xu, H., Chanchevalap, S., Shen, W., Wu, J., Li, Y., and Jiang, C. (2000) J. Biol. Chem. 275, 31573–31580[Abstract/Free Full Text]
  29. Leung, Y. M., Zeng, W. Z., Liou, H. H., Solaro, C. R., and Huang, C. L. (2000) J. Biol. Chem. 275, 10182–10189[Abstract/Free Full Text]
  30. Yang, Z., Xu, H., Cui, N., Qu, Z., Chanchevalap, S., Shen, W., and Jiang, C. (2000) J. Gen. Physiol. 116, 33–45[Abstract/Free Full Text]
  31. Schulze, D., Krauter, T., Fritzebschaft, H., Soom, M., and Baukrowitz, T. (2003) J. Biol. Chem. 278, 10500–10505[Abstract/Free Full Text]
  32. Henry, P., Pearson, W. L., and Nichols, C. G. (1996) J. Physiol. 495, 681–688[Medline] [Order article via Infotrieve]
  33. Zhu, G., Qu, Z., Cui, N., and Jiang, C. (1999) J. Biol. Chem. 274, 11643–11646[Abstract/Free Full Text]
  34. Ho, I. H., and Murrell-Lagnado, R. D. (1999) J. Physiol. 520, 645–651[Abstract/Free Full Text]
  35. Chan, K. W., Sui, J. L., Vivaudou, M., and Logothetis, D. E. (1997) J. Biol. Chem. 272, 6548–6555[Abstract/Free Full Text]
  36. Vivaudou, M., Chan, K. W., Sui, J. L., Jan, L. Y., Reuveny, E., and Logothetis, D. E. (1997) J. Biol. Chem. 272, 31553–31560[Abstract/Free Full Text]
  37. Gribble, F. M., Ashfield, R., {Delta}mmälä, C., and Ashcroft, F. M. (1997) J. Physiol. 498, 87–98[CrossRef][Medline] [Order article via Infotrieve]
  38. Maduh, E. U., Borowitz, J. L., and Isom, G. E. (1990) Toxicol. Appl. Pharmacol. 106, 201–208[CrossRef][Medline] [Order article via Infotrieve]
  39. Collins, A., and Larson, M. (2002) J. Biol. Chem. 277, 35815–35818[Abstract/Free Full Text]
  40. Qu, Z. Q., Zhu, G. Y., Yang, J., Cui, N. R., Li, Y., Chanchevalap, S., Sulaiman, S., Haynie, H., and Jiang, C. (1999) J. Biol. Chem. 274, 13783–13789[Abstract/Free Full Text]
  41. Karle, C. A., Zitron, E., Zhang, W., Wendt-Nordahl, G., Kathöfer, S., Thomas, D., Gut, B., Scholz, E., Vahl, CF., Katus, H., and Kiehn, J. (2002) Circulation 106, 1493–1499[Abstract/Free Full Text]
  42. Hill, J. J., and Peralta, E. G. (2001) J. Biol. Chem. 276, 5505–5510[Abstract/Free Full Text]
  43. Baukrowitz, T., Schulte, U., Oliver, D., Herlitze, S., Krauter, T., Tucker, S. J., Ruppersberg, J. P., and Fakler, B. (1998) Science 282, 1141–1144[Abstract/Free Full Text]
  44. Shyng, S. L., Cukras, C. A., Harwood, J., and Nichols, C. G. (2000) J. Gen. Physiol. 116, 599–608[Abstract/Free Full Text]
  45. Soom, M., Schonherr, R., Kubo, Y., Kirsh, C., Klinger, R., and Heinemann, S. H. (2001) FEBS Lett. 490, 49–53[CrossRef][Medline] [Order article via Infotrieve]
  46. Nishida, M., and MacKinnon, R. (2002) Cell 111, 957–965[CrossRef][Medline] [Order article via Infotrieve]
  47. Rhee, J. S., Betz, A., Pyott, S., Reim, K., Varoqueaux, F., Augustin, I., Hesse, D., Thomas, C., Sudhof, T. C., Takahashi, M., Rosenmund, C., and Brose, N. (2002) Cell 108, 121–133[CrossRef][Medline] [Order article via Infotrieve]
  48. Schechtman, D., and Mochly-Rosen, D. (2001) Oncogene 20, 6339–6347[CrossRef][Medline] [Order article via Infotrieve]
  49. Lopatin, A. N., Makhina, E. N., and Nichols, C. G. (1994) Nature 372, 366–369[CrossRef][Medline] [Order article via Infotrieve]
  50. Xu, H. X., Cui, N. R., Yang, Z. J., Wu, J. P., Giwa, L. R., Abdulkadir, L., Sharma, P., and Jiang, C. (2001) J. Biol. Chem. 276, 12898–12902[Abstract/Free Full Text]
  51. Zeng, W. Z., Li, X. J., Hilgemann, D. W., and Huang, C. L. (2003) J. Biol. Chem. 278, 16852–16856[Abstract/Free Full Text]
  52. Zhang, L. Y., Lee, J. K., John, S. C., Uozumi, N., and Kodama, I. (2004) J. Biol. Chem. 279, 7037–7047[Abstract/Free Full Text]

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