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Am J Physiol Cell Physiol 290: C345-C351, 2006; doi:10.1152/ajpcell.00091.2005
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Voltage-gated potassium channel Kv1.3 regulates GLUT4 trafficking to the plasma membrane via a Ca2+-dependent mechanism

Yanyan Li,1 Peili Wang,1 Jianchao Xu,1,2 and Gary V. Desir1,2

1Department of Medicine, Yale University School of Medicine, New Haven; and 2Veterans Administration Connecticut Health System, West Haven, Connecticut

Submitted 3 March 2005 ; accepted in final form 9 August 2005


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
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Kv1.3 is a voltage-gated K+ channel expressed in insulin-sensitive tissues. We previously showed that gene inactivation or pharmacological inhibition of Kv1.3 channel activity increased peripheral insulin sensitivity independently of body weight by augmenting the amount of GLUT4 at the plasma membrane. In the present study, we further examined the effect Kv1.3 on GLUT4 trafficking and tested whether it occurred via an insulin-dependent pathway. We found that Kv1.3 inhibition by margatoxin (MgTX) stimulated glucose uptake in adipose tissue and skeletal muscle and that the effect of MgTX on glucose transport was additive to that of insulin. Furthermore, whereas the increase in uptake was wortmannin insensitive, it was completely inhibited by dantrolene, a blocker of Ca2+ release from intracellular Ca2+ stores. In white adipocytes in primary culture, channel inhibition by Psora-4 increased GLUT4 translocation to the plasma membrane. In these cells, GLUT4 protein translocation was unaffected by the addition of wortmannin but was significantly inhibited by dantrolene. Channel inhibition depolarized the membrane voltage and led to sustained, dantrolene-sensitive oscillations in intracellular Ca2+ concentration. These results indicate that the apparent increase in insulin sensitivity observed in association with inhibition of Kv1.3 channel activity is mediated by an increase in GLUT4 protein at the plasma membrane, which occurs largely through a Ca2+-dependent process.

insulin; glucose; diabetes; calcium

VOLTAGE-GATED POTASSIUM (Kv) channels regulate cell membrane potential (Vm) by controlling the rate of K+ exit from the cell and can therefore modulate a large number of cellular processes. Kv1.3 is a Shaker-related Kv channel that is expressed in several tissues, including insulin-sensitive tissues such as fat and skeletal muscle. The channel has been implicated in the regulation of a variety of physiological functions, including cell volume regulation, apoptosis, T-cell activation, and renal solute homeostasis. Channel activity is controlled in a complex manner, not only by changes in Vm but also by threonine, serine, and tyrosine phosphorylation. Serotonin and insulin can both downregulate the activity of the Kv1.3 channel. In the case of insulin, channel inhibition has been observed in the olfactory bulb and is mediated by phosphorylation of multiple tyrosine residues (7).

Despite extensive data regarding the kinetic and pharmacological properties as well as the regulation of Kv1.3, its physiological role is not totally understood. Examination of Kv1.3-knockout mice (Kv1.3–/–) generated by gene targeting revealed a previously unrecognized role for Kv1.3 in body weight regulation (25). Indeed, Kv1.3–/– mice weigh significantly less than their control littermates. Moreover, they are protected from diet-induced obesity and gain significantly less weight than their littermate controls when fed a high-fat diet. Although food intake did not differ significantly between Kv1.3–/– mice and controls, basal metabolic rate measured at rest using indirect calorimetry was significantly higher in the knockout animals. These data indicate that Kv1.3 channels may participate in the pathways that regulate body weight as well as that channel inhibition increases basal metabolic rate.

Furthermore, we previously showed that acute inhibition (2 h) of Kv1.3 channel activity in wild-type, obese, and diabetic mice increased insulin sensitivity independently of body weight (26). Baseline and insulin-stimulated glucose uptake are increased in adipose tissue and skeletal muscle of Kv1.3–/– mice compared with weight-matched control mice. Inhibition of Kv1.3 activity in wild-type mice suppresses JNK activity in fat and skeletal muscle, decreases IL-6 and TNF-{alpha} secretion, and facilitates the translocation of glucose transporter 4 (GLUT4) to the plasma membrane.

Recent studies have indicated that Kv1.3 might serve as a substrate of insulin receptors (IRs). Kv1.3 channel activity is inhibited by tyrosine phosphorylation both in vivo and in vitro (7). Therefore, it is possible that Kv1.3 increases peripheral glucose uptake by augmenting the effect of insulin. Alternatively, Kv1.3 inhibition may depolarize the cell membrane and facilitate peripheral glucose uptake via insulin-independent mechanisms. Such a pathway has been identified in skeletal muscle, where either muscle contraction or Vm depolarization induced by high extracellular K+ concentration appears to stimulate glucose uptake by recruiting GLUT4 to the plasma membrane in the absence of insulin.

In this study, we further examined the effect Kv1.3 inhibition on GLUT4 trafficking and tested whether channel inhibition stimulates glucose uptake and GLUT4 translocation by augmenting the action of insulin or by acting through an insulin-dependent pathway.


   METHODS
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RESULTS
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Glucose uptake in adipose tissue. Baseline and insulin-stimulated glucose uptake were measured in epididymal fat isolated from wild-type mice as described previously (26). All protocols that involved the use of animals were reviewed and approved by the Animal Committee at Veterans Administration Connecticut Health System.

Western blot analysis. Homogenates were prepared from the white fat of Kv1.3+/+ mice and 3T3-L1 cells. Protein (10 µg) was resolved when 10% SDS-PAGE was performed, and then the protein was transferred onto a nitrocellulose membrane, which was probed with a goat anti-human Kv1.3 PAb (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive protein bands were visualized using ECL (PerkinElmer, Boston, MA) as previously described (28).

Immunodetection of GLUT4 and Kv1.3 in adipocytes. Primary cultures of epididymal fat isolated from wild-type mice under sterile conditions were established as described previously (26) using the method of Cabrero et al. (3). Cells were treated with the compounds of interest for 30 min at 37°C and then fixed with 3% paraformaldehyde in PBS for 3 min at room temperature. GLUT4 expression was detected in nonpermeabilized adipocytes using immunofluorescence (anti-GLUT4 antibody directed against an external epitope; Santa Cruz Biotechnology). Kv1.3 expression was measured in permeabilized cells using a goat anti-human Kv1.3 PAb (1:200 dilution; Santa Cruz Biotechnology). The Texas red-conjugated secondary antibody was obtained from Vector Laboratories (Burlingame, CA). Excess antibody was removed by washing the cells three times with PBS, and then the cells were mounted onto glass microscope slides and viewed using a Zeiss LSM 510 confocal imaging system.

Vm measurement. NIH-3T3 cells were cultured on glass coverslips and incubated for 20 min with the voltage-sensitive fluorescent dye pyridinium, 4-{2-[6-(dibutylamino)-2-naphthalenyl]ethenyl}-1-(3-sulfopropyl)-hydroxide (di-4-ANEPPS; Molecular Probes, Eugene, OR), at a final concentration 10 µM. The cells were washed three times in Krebs-Ringer bicarbonate (KRB) buffer. Images of signal were obtained before and after the addition of 5-(4-phenylbutoxy)psoralen (Psora-4; 2.5 nM) using a FluoView confocal laser-scanning microscope (IX70; Olympus, Melville, NY) with a UPlan Fluorite N x20 magnification lens objective (numerical aperture 0.5). Images were scanned at a 0.33 s/frame rate. The data were analyzed using FluoView image-processing software (version 2.1) and saved to a spreadsheet software file (Excel 2000, Microsoft, Redmond, WA).

Measurement of intracellular Ca2+ concentration. NIH-3T3 adipocytes were incubated for 15 min in KRB buffer with 5 µM fluo-3 AM (Molecular Probes). The cells were then washed three times in KRB buffer. For some studies, the cells were incubated and washed in Ca2+-free KRB buffer. The cells were scanned before and after the addition of the drugs of interest using a FluoView confocal laser-scanning microscope with a UPlan Fluorite N x20 magnification lens objective (numerical aperture 0.5). Images were obtained at a 0.3 s/frame rate, and 30 frames were obtained to record a total of 9 s for each condition. The data were analyzed using FluoView image-processing software (version 2.1) and saved to a spreadsheet software file (Excel 2000; Microsoft).

Statistical analysis. The two-tailed Student’s t-test for unpaired samples was used to analyze the data shown, and data for which P < 0.05 are indicated as appropriate.


   RESULTS
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DISCUSSION
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Kv1.3 protein expression was confirmed in adipocytes using Western blot analysis of white fat isolated from wild-type mice. A single band of the expected size (68 kDa) was detected (Fig. 1A), and its specificity was confirmed by its absence in white fat obtained from Kv1.3–/– mice (Fig. 1A). Kv1.3 protein expression was also detected in the NIH-3T3 adipocyte cell line (Fig. 1A). The expression level in adipose tissue was greater than that in NIH-3T3 cells. Confocal microscopy of permeabilized adipocytes revealed that although Kv1.3 was expressed at the plasma membrane (Fig. 1B), a significant fraction of Kv1.3 protein appeared to be intracellular.



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  		Fig. 1. Voltage-gated K+ channel 1.3 (Kv1.3) expression in adipocytes. A: Kv1.3 expression in 3T3-L1 adipocytes. Protein expression was assayed using Western blot analysis, and a band of the expected size (68–72 kDa) was detected in adipose tissue of wild-type mice and in 3T3-L1 adipocytes. The signal was absent from Kv1.3–/– adipocytes. B: confocal images showing subcellular localization of adipocytes in primary culture. Kv1.3 was detected at the plasma membrane and throughout the cytoplasm.

 
We previously showed that baseline glucose uptake in skeletal muscle and adipose tissue was higher in Kv1.3–/– mice than in wild-type mice. Furthermore, Kv1.3 inhibition with margatoxin (MgTX) significantly increased glucose uptake in these tissues. We thus asked whether the Kv1.3-dependent stimulation of glucose uptake is mediated by an insulin-regulated pathway. Because insulin-dependent glucose uptake involves the activation of class 1A phosphatidylinositol 3'-kinase (PI3K), we reasoned that if the action of Kv1.3 on glucose uptake is insulin independent, PI3K inhibition should have no significant effect on the observed transport rates. Adipose tissue was isolated from wild-type mice, and glucose uptake was assayed under various conditions. We found that 1 nM insulin increased glucose uptake 1.9-fold (n = 3), whereas 10 and 100 nM insulin caused 4.6-fold (n = 5) and 4.5-fold increases (n = 5), respectively. Therefore, 10 nM insulin was used in all subsequent studies. Because MgTX inhibits Kv1.3 with a Ki of 50–100 pM, the dose of 1 nM was chosen to maximize the specific inhibition of Kv1.3 channels. As shown in Fig. 2A, we confirmed that either dantrolene, which inhibits Ca2+ release from intracellular stores, or wortmannin (PI3K inhibitor) alone had no effect on baseline glucose uptake and that insulin-stimulated glucose transport was highly sensitive to inhibition by wortmannin and was partially blocked by dantrolene. In contrast, MgTX-stimulated glucose uptake in adipose tissue was unaffected by the addition of wortmannin, whereas dantrolene completely abrogated the stimulatory effect of MgTX on glucose transport (Fig. 2B). These data support the notion that Kv1.3 inhibition increases glucose uptake via an PI3K-independent process. Furthermore, they suggest a critical role for the release of Ca2+ from intracellular stores.



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  		Fig. 2. Kv1.3-mediated glucose uptake is phosphatidylinositol 3'-kinase independent. A: effect of insulin on glucose uptake in adipose tissue. Baseline glucose uptake was measured at 20 min in PBS. Insulin (10 nM) significantly increased baseline uptake (n = 5). Wortmannin (100 nM) blocked the effect of insulin completely (n = 5), whereas dantrolene (DAN; 12.5 µM) was a partial inhibitor (n = 5). B: effect of margatoxin (MgTX) on glucose uptake. Baseline glucose uptake measured at 20 min in PBS was significantly stimulated by MgTX (1 nM) (n = 5). DAN (12.5 µM) blocked the effect of MgTX completely (n = 5), whereas wortmannin (100 nM) had no effect (n = 5). The action of insulin and MgTX was additive (n = 5).

 
Insulin stimulates glucose uptake by causing the glucose transporter GLUT4 to translocate from intracellular stores to the plasma membrane. We previously showed that Kv1.3 inhibition also increases the amount of GLUT4 protein at the plasma membrane in both adipocytes and skeletal muscle (26). Because Kv1.3-mediated glucose transport is dantrolene sensitive, the Ca2+ dependence of GLUT4 translocation was examined in untransfected, nonpermeabilized adipocytes in primary culture using a GLUT4 antibody raised against an extracellular epitope. Psora-4, the most potent small-molecule inhibitor of Kv1.3 (23), caused a large increase in plasma membrane GLUT4 in the absence of external Ca2+ (Fig. 3, far and middle left images). As noted in the transport studies, Kv1.3’s action on GLUT4 translocation was insensitive to wortmannin but almost completely inhibited by dantrolene. These data indicate that Kv1.3 inhibition stimulates glucose uptake by facilitating the translocation of GLUT4 to the plasma membrane through a mechanism that is critically dependent on Ca2+ release from intracellular stores.



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  		Fig. 3. DAN inhibits MgTX-stimulated glucose transporter 4 (GLUT4) translocation in adipocytes. GLUT4 was detected in nonpermeabilized NIH-3T3 adipocytes using confocal immunofluorescence microscopy with an anti-GLUT4 antibody directed against an extracellular epitope (n = 5, representative studies shown). Far left: baseline measurement with cells incubated in PBS with 2 mM CaCl and 5 mM EGTA; middle left: Kv1.3 inhibition with 5-(4-phenylbutoxy)psoralen (Psora-4; 2.5 nM); middle right: Kv1.3 inhibition with Psora-4 (2.5 nM) and DAN (12.5 µM); far right: Kv1.3 inhibition with Psora-4 (2.5 nM) and wortmannin (100 nM).

 
Kv1.3 is a voltage-gated K+ channel (activation threshold of approximately –30 mV) (27) that is known to regulate Vm and Ca2+ signaling in lymphocytes (4). Kv1.3 inhibition is associated with a decrease in intracellular Ca2+ in T lymphocytes because the channel protects against Vm depolarization and maintains a favorable electrochemical gradient for Ca2+ entry. The role of Kv1.3 in adipocyte Ca2+ signaling was examined. As shown in Fig. 4A, Kv1.3 inhibition in adipocytes led to repeated depolarization spikes, suggesting that the channel plays an important role in maintaining baseline Vm. Surprisingly, Kv1.3 inhibition was associated with a significant increase in intracellular Ca2+ concentration and the generation of Ca2+ waves (Fig. 4B), which were both completely inhibited by dantrolene.



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  		Fig. 4. Kv1.3 inhibition depolarizes membrane voltage (Vm) and increases cell Ca2+. A: Vm measurement. Changes in Vm were monitored in NIH-3T3 adipocytes using 4-{2-[6-(dibutylamino)-2-naphthalenyl]ethenyl}-1-(3-sulfopropyl)-hydroxide. Arrow indicates addition of 2.5 nM Psora-4. Increased in fluorescence signifies Vm depolarization. B: intracellular Ca2+ concentration measurement. Changes in intracellular Ca2+ concentration were monitored in NIH-3T3 adipocytes (n = 8) using fluo-3 AM. Arrow indicates the addition of Psora-4 (2.5 nM). Increased fluorescence denotes a rise in intracellular Ca2+ concentration. Some cells were preincubated with DAN (12.5 µM), and the signal obtained from these cells is indicated by the bold line. A representative signal is depicted for each condition.

 
To test whether Vm depolarization and Ca2+ release from intracellular stores were critical mechanisms through which Kv1.3 inhibition enhanced GLUT4 translocation, adipocytes were depolarized by raising external K+ concentration. As shown in Fig. 5 (left), compared with 5 mM K+ (Vm approximately –60 mV), both 50 mM K+ (Vm approximately –15 mV) and 150 mM K+ (Vm ~0 mV) increased GLUT4 translocation to the plasma membrane in the absence of external Ca2+. The effect of Vm depolarization on GLUT4 traffic was almost completely inhibited by dantrolene, which inhibits Ca2+ release from intracellular stores (Fig. 5, right). These data strongly support the notion that depolarization-induced GLUT4 translocation does not require external Ca2+ but is dependent on Ca2+ release from intracellular compartments.



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  		Fig. 5. Membrane depolarization stimulates GLUT4 translocation. GLUT4 was detected in permeabilized adipocytes in primary culture using confocal immunofluorescence microscopy with an anti-GLUT4 antibody (n = 3, representative studies shown). Left: baseline measurement with cells incubated in buffer containing the indicated intracellular K+ concentration ([K]), osmolality was maintained by reducing intracellular Na+ concentration; right: effect of DAN (12.5 µM) on depolarization-mediated GLUT4 translocation.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Kv1.3 is a novel component of the pathways that regulate body weight, energy expenditure, and glucose homeostasis (25, 26). Its effect on glucose metabolism is independent of body weight, which is evident in obese and diabetic mice. Kv1.3 channel inhibition increases peripheral insulin sensitivity.

Two major pathways appear to mediate glucose uptake in tissues that regulate glucose homeostasis. The first has been studied extensively and involves the binding of insulin to its receptor (a receptor tyrosine kinase IR) present at the plasma membrane of cells of insulin-responsive tissues, including skeletal muscle, fat, and liver (10, 16, 19). When insulin binds to its receptor, the IR undergoes a series of conformational changes leading to autophosphorylation and the initiation of a complex cascade of intracellular signaling events, including the tyrosine phosphorylation of a number of downstream targets, ultimately leading to the translocation of GLUT4 to the plasma membrane and to increased glucose uptake. The PI3K and c-Cbl-associated protein (CAP)-Cbl pathways are critical to the action of insulin on glucose transport (20).

PI3K plays a central role in the cellular action of insulin (16, 19). The complex is composed of a catalytic subunit (p110) and a regulatory subunit (p85) and interacts with IRs via its Src homology 2 domains. PI3K has serine-threonine kinase activity and increases the generation of phosphatidylinositol 3-phosphate (PIP3). PIP3 then activates PKB, PKC-{zeta}, and PKC-{lambda}. The CAP-Cbl pathways represent the other important mechanism involved in insulin-mediated glucose uptake. Cbl is a protooncogene that is phosphorylated at tyrosine residues by IRs. Phosphorylated Cbl binds to the adapter protein CAP and translocates to lipid rafts, where it interacts with flotillin. Ultimately, this interaction leads to the activation of TC10, a G protein. The presence of activated TC10 in lipid rafts is critical for the translocation of GLUT4 to the plasma membrane. Our data indicate that Kv1.3 stimulated glucose transport via a PI3K-independent mechanism; however, they do not exclude the possibility that Kv1.3 acted on the insulin-dependent pathway at a step distal to PI3K or through the CAP-Cbl pathway. Interestingly, Kv1.3 localizes to lipid rafts in T lymphocytes, where it forms a complex with CD95 and p56Lck (2, 21). Whether Kv1.3 also traffics to lipid rafts in adipocytes is not yet known.

The second major mechanism required for glucose uptake is evident during muscular contraction, when GLUT4 translocates to the cell surface and increases cellular glucose uptake in an insulin-independent manner. Indeed, the effects of insulin and exercise on GLUT4 translocation are additive in skeletal muscle. Wortmannin, an agent that inhibits PI3K activity, blocks insulin-stimulated GLUT4 translocation in skeletal muscle and cultured L6 myotubes but has no effect on contraction-mediated or Ca2+-dependent GLUT4 translocation in skeletal muscle (1, 9). Under certain pathophysiological conditions, such as diabetes, insulin resistance, or obesity, in which insulin-stimulated GLUT4 translocation is impaired, contraction-stimulated GLUT4 translocation in skeletal muscle is maintained (6, 14). Contraction-mediated GLUT4 translocation is Ca2+ dependent, but insulin-dependent stimulation proceeds independently of Ca2+ concentration in both skeletal muscle and cardiac myocytes. It is noteworthy that at least in adipocytes, intracellular Ca2+ may also play an important role in insulin-stimulated glucose transport (24). Finally, vesicle density and sedimentation velocity data suggest that insulin and muscle contraction mobilize distinct intracellular GLUT4 vesicles (5).

The precise mechanisms that underlie the effect of muscle contraction on glucose transport are not well defined (8, 13, 17, 22). It is currently thought that muscle contraction increases glucose uptake via two distinct pathways (17, 18). Cell Vm depolarization, which initiates muscle contraction, causes a rise in intracellular Ca2+ concentration and leads to the activation of PKC and other proteins. Factors that affect resting cell Vm, such as Kv channel activity (Kv1.3 and other Kv channels), could modulate intracellular Ca2+ concentration and regulate glucose uptake. The other important pathway relates to the cellular stress induced by contraction with subsequent changes in the levels of ATP, glycogen, and oxygen. This pathway is postulated to involve AMPK, a putative sensor of cellular fuel stores, and nitric oxide synthase.

Inhibition of Kv1.3 by Psora-4 initially caused Vm depolarization and resulted in Vm oscillations. We speculate that these oscillations arose from the interaction of Ca2+-activated K+ channels, inward rectifiers, and Cl– channels. Indeed, Kv1.3, along with an inward rectifier and a swelling-activated Cl– current, was recently shown to participate in the regulation of baseline and dynamic Vm in cultured rat microglia (15). Ca2+ oscillations were also noted with Kv1.3 inhibition. Of note is that depolarization-evoked Ca2+ release and oscillations from intracellular stores have been observed in nonexcitable cells such as rat megakaryocytes (11) and pheochromocytoma (PC)-12 neurosecretory cells (12). The precise mechanism that underlies this phenomenon still is unclear; however, in megakaryocytes, it is independent of external Ca2+ and of the activity of the Na+/Ca2+ exchanger but appears to require an active inositol 1,4,5-trisphosphate receptor. In PC-12 cells, Vm depolarization is accompanied by intracellular acidification, which is postulated to stimulate a H+-Ca2+ antiporter present in the secretory vesicles. The fact that Vm depolarization achieved using high external K+ (150 mM, Vm ~0 mV) also stimulated GLUT4 translocation suggests that depolarization-induced Ca2+ release from intracellular stores is a critical factor.

Kv1.3 inhibition in adipocytes leads to Vm depolarization, Ca2+ release from intracellular stores, increased intracellular Ca2+ concentration, and ultimately GLUT4 translocation (Fig. 6). The exact molecular mechanisms underlying the action of Ca2+ on GLUT4 translocation are currently unknown. It should be noted that Kv1.3’s role in intracellular Ca2+ signaling of adipocytes is quite distinct from that which it plays in resting lymphocytes. In these cells, Kv1.3 inhibition decreases Ca2+ entry (4). We conclude that Kv1.3 modulates glucose transport through a PI3K-independent pathway that requires Ca2+ release from intracellular stores.



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  		Fig. 6. Scheme showing the cellular mechanism of increased glucose uptake in adipocytes with Kv1.3 inhibition. See text for details.

 

   GRANTS
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REFERENCES
 
This work was supported by a Department of Veterans Affairs Merit Review Award through National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants DK-48105B and DK-065172 and by NIDDK Grants DK-0291702 and DK-064317 (to G. V. Desir).


   ACKNOWLEDGMENTS
 
We thank Drs. George K. Chandy and Heike Wulff for providing the Kv1.3 inhibitor Psora-4.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. V. Desir, Section of Nephrology, Dept. of Medicine, Yale School of Medicine, 333 Cedar St., LMP 2073, PO Box 208029, New Haven, CT 06520-8029 (e-mail: gary.desir{at}yale.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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28. Yao X, Tian S, Chan HY, Biemesderfer D, and Desir G. Expression of KCNA10, a voltage-gated K channel, in glomerular endothelium and at the apical membrane of the renal proximal tubule. J Am Soc Nephrol 13: 2831–2839, 2002.[Abstract/Free Full Text]




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