Expression of KCNA10, a Voltage-Gated K Channel, in Glomerular Endothelium and at the Apical Membrane of the Renal Proximal Tubule
Xiaoqiang Yao,
Shulan Tian*,
Ho-Yu Chan,
Daniel Biemesderfer* and
Gary V. Desir*,
*Yale University School of Medicine; WHVA Medical Center, New Haven, Connecticult; and Chinese University of Hong Kong, Hong Kong.
Correspondence to Dr. Gary V. Desir, Department of Medicine, Section of Nephrology, Yale University School of Medicine, West Haven VA Medical Center, 333 Cedar Street, LMP 2073, P.O. Box 208029, New Haven, CT 06520-8029. Phone: 203-506-2500; Fax: 508-462-8950;E-mail: gary.desir{at}yale.edu
ABSTRACT. Potassium (K) channels regulate cell membrane potentialand modulate a number of important cellular functions. KCNA10is a cyclic nucleotide-gated, voltage-activated K channel thatis detected in kidney, heart, and aorta by Northern blot andpostulated to participate in renal K metabolism and to regulatevascular tone. The aim of this study was to establish the cellularand subcellular localization of KCNA10 in kidney and vasculartissues. An anti-KCNA10 polyclonal antibody was generated, andimmunocytochemical studies were performed on rat kidney. KCNA10protein was easily detectable at the apical membrane of ratproximal tubular cells, and a weaker signal was also evidentin the glomerulus. In situ hybridization experiments confirmedthe immunocytochemical studies and revealed KCNA10 expressionin human proximal tubular cells, glomerular and vascular endothelialcells, and also in vascular smooth muscle cells. The data suggestthat KCNA10 may facilitate proximal tubular sodium absorptionby stabilizing cell membrane voltage. Furthermore, its presencein endothelial and vascular smooth muscle cells supports thenotion that it also regulates vascular tone.
Potassium (K) channels are membrane proteins that participatein many cellular processes primarily by regulating membranepotential. KCNA10 is a voltage-gated K (Kv) channel gene relatedto the Shaker superfamily (1), and its most distinguishing featurerelates to the presence of a putative cyclic nucleotide-binding(CNB) domain at the carboxy terminus. A few other K channelsalso contain CNB domains and may belong to new subclass of Kchannels with structural features common to both Kv channelsand to cyclic nucleotide-gated cation channels (2–5).For instance, the HERG channel binds cAMP, regulates cardiacrepolarization, and is mutated in some cases of long QT syndrome(6,7). Other potential physiologic roles for these proteinsinclude arterial vasorelaxation (8), hormone secretion (9,10),and neuronal membrane excitability (11).
The subunit of KCNA10 (KCNA10) (1) mediates voltage-gated Kcurrents that exhibit minimal steady-state inactivation. Channelsare activated by depolarizations more positive than -66 mV.The channel displays an unusual inhibitor profile, because inaddition to being blocked by classical K channel blockers, itis also sensitive to inhibitors of cyclic nucleotide gated cationchannel such as verapamil and pimozide. Tail current analysisindicates that KCNA10 has a potassium-to-sodium selectivityratio of at least 15:1. The single channel conductance is approximately11 pS, and channel activity is inhibited by protein kinase C.
Numerous studies have shown that the properties observed forcloned subunits very rarely match currents known to exist innative tissues (12). Most often it is because the subunitsinteract with accessory proteins that modify their kinetic andpharmacologic properties. We recently identified an accessorysubunit (KCNA4B) of KCNA10 by yeast-2-hybrid using the C terminusof the KCNA10 as bait. KCNA4B binds to the C terminus of KCNA10and can be immunoprecipitated in vitro and in vivo. The biologicsignificance of this interaction is reflected in the fact thatKCNA4B increases KCNA10 current expression by at least 2.8-foldand also alters its sensitivity to cAMP.
In previous studies, we documented KCNA10 expression by Northernblotting and PCR in renal tubular cells and blood vessels vasculartissue (13,14). PCR of dissected nephron segments suggestedwidespread expression of KCNA10 because a signal was observedin all nephron segments tested, including glomeruli, proximaltubule, connecting tubule cortical collecting ducts, and renalvessels. There were three significant limitations of the PCRanalysis. First, the primers used for analysis resided on themain exon of KCNA10, and we could not conclusively rule outgenomic contamination. Second, we could not exclude the possibilitythat dissected tubule segments were contaminated by other tissues,including blood vessels. Lastly, PCR analysis could not ascertainif the protein was actually expressed and if expression wasbasolateral or apical. The task of ascribing a definitive functionalrole to KCNA10 will be greatly facilitated by accurate and preciseknowledge of channel expression at the nephron segment, cellular,and membrane level; we therefore generated an anti-KCNA10 polyclonalantibody and used it to localize KCNA10 in the kidney.
Antibody Preparation
Polyclonal sera were raised in rabbits by Biosynthesis Inc.(Lewisville, TX) against a peptide corresponding to amino acids47 to 65 (SNWRVLISDNTNHETAFSK) of mouse KCNA10 (mKCNA10). Thesera were purified before use by affinity chromatography usinga column containing the mKCNA10 peptide.
In Vitro Translation
The coding regions of hKCNA10 (accession No. U96110) and hKv1.3(accession No. XM084080) were amplified by PCR with a proofreadingpolymerase and sense primers containing a T7 polymerase site.The PCR products were used as template for cRNA synthesis usingT7 polymerase (Ambion), and in vitro translation was carriedout for each individual cRNA using rabbit reticulocyte lysates(Ambion).
Preparation of Renal Membrane Vesicles
Adult male Harlan Sprague-Dawley rats (Charles River) were sacrificedby injection of sodium pentobarbital (Butler Co., Columbus,OH), and microvillus membrane vesicles were prepared from renalcortex using the MgCl2 precipitation method as described previously(15).
Western Blot Analyses
Protein samples were solubilized in SDS-PAGE sample buffer andseparated by SDS-PAGE using 7.5% polyacrylamide gels. For immunoblotting,proteins were transferred to polyvinylidene difluoride (MilliporeImmobilon-P) at 500 mA for approximately 10 h at 4°C witha TransphorTM transfer electrophoresis unit (Hoefer ScientificInstruments, San Francisco, CA). The blot was stained with PonceauS in 0.5% trichloroacetic acid, and nonspecific binding wasblocked by incubation in Blotto (5% nonfat dry milk in phosphate-bufferedsaline [PBS], pH 7.4) for 1 to 3 h. Blots were then incubatedfor 1 to 5 h with anti-KCNA10, the primary antibody, dilutedin Blotto, at dilutions ranging from 1:200 to 1:1000. In somestudies, (peptide competition), the primary antibody was preincubatedwith 100 ng of the antigenic peptide for 1 h before it was addedto the blot. The blots were then washed in Blotto and incubatedfor 1 h with an appropriate horseradish peroxidase-conjugatedsecondary antibody diluted 1:2000 in Blotto. The blots werethan thrice washed: once with Blotto, once in PBS (pH 7.4),and once in distilled water. Bound antibody was detected byECLT chemiluminescence system (Amersham Pharmacia Biotech) accordingto the manufacturer’s protocols.
Immunolocalization
For light microscopic studies, Epon sections (0.5 mm) are cutwith a glass knife, stained with toluidine blue, and examinedwith a Zeiss Axiophot microscope. Fixed tissue was embeddedin paraffin, and 0.5- to 2.0-mm-thick sections were cut usinga glass knife mounted on the sectioning stage of a Reichertultramicrotome. Sections were then mounted on glass coverslips,deparaffinized using xylene, and rehydrated in graded ethanolsand PBS. Sections were microwaved in buffer containing 10 mMcitrate in Tris-buffered saline (TBS) at 40% power for 20 min.After washing in TBS, the sections were further denatured using1% SDS in TBS for 5 min. After washing in TBS, the sectionswere immunolabeled with the primary antibody at a dilution of1:100 or with the preimmune serum (1:100 dilution). They werewashed in blocking solution and then incubated for 1 h at roomtemperature with an FITC-labeled goat, anti-rabbit secondaryantibody (Zymed Laboratories, South San Francisco, CA) at adilution of 1:100. Finally, the slides were washed in PBS, preservedin crystal/mount (Biomeda, Foster City, CA), and examined within4 h using a Zeiss fluorescence microscope. Representative pictureswere recorded and stored digitally.
In Situ Hybridization
A 334-bp fragment of KCNA10 (1), -4 to +330, (GenBank accessionNo. U96110; start Codon counted as position 1) was amplifiedby PCR using the following primers: sense 5'-TAGAATGGATGTGTGTGGCTGGAA-3'and antisense 5'-GTCTCTGGGAACTGACTAAGGGTT-3'. That region ishighly specific for KCNA10, and a GenBank search revealed nosignificant homology to other known genes. The amplified productwas cloned into pPCR-ScriptAmp cloning vector (Stratagene),and the authenticity of the clone was confirmed by automaticDNA sequencing using ABI 310 autosequencer (Perkin Elmer). Theclone was made linear by digestion with NotI, and a DIG-labeledantisense riboprobe was synthesized with T7 RNA polymerase usinga DIG-labeling kit (Roche Biochemicals, Germany). The senseribopobe was synthesized by digesting with EcoRI and transcribingwith T3 RNA polymerase. An adult human kidney and cerebral arterywere obtained from an autopsy case with written consent of familymembers under an approved protocol at the Chinese Universityof Hong Kong. Tissues were fixed overnight with 4% paraformaldehydein PBS. We examined tissues from two deceased subjects whosebodies were immediately transferred to cold room and kept a4°C. Autopsy was done within 5 to 7 h. There were no differenceswith regards to KCNA10 expression. The data presented in theFigures were obtained from the tissues of a 59-yr-old womanwho died of cardiac tamponade caused by a dissecting aorticaneurysm. Her kidneys showed only mild, age-related nephrosclerosis,and she had no history of hypertension. The protocol that followsis a modification of that of Yew et al. (16). The tissues weredehydrated through graded ethanol, cleared with xylene, embeddedin paraffin, and cut into 6-µm-thick sections. The sectionswere washed briefly with diethylpyrocarbonate-treated waterfollowed by PBS for 10 min. They were then digested with proteinaseK (10 µg/ml) at 37°C for 10 min. Hybridization wasperformed at 65°C in buffer containing 4x SSC, 10% dextransulfate, 1x Denhardt solution, 5 mM EDTA, 0.1% Chaps, 50% deionizedformamide, 200 µg/ml herring sperm DNA, 200 ng/ml DIG-labeledprobe. The slides were then washed four times for 15 min eachin 2x SSC and 0.1% SDS at 65° and then thrice in 0.2x SSCand 0.1% SDS at 65°C and thrice with 0.2x SSC at room temperature.Colorimetric detection was performed using an anti-DIG antibodyconjugated to alkaline phosphatase followed by incubation withNBT/BCIP color substrates using a digoxogenin-nucleic acid detectionkit (Roche, Germany) as described previously. The results wereassessed by light microscopy and digitally recorded. Positivesignals appeared within 2 to 3 min of incubation with NBT/BCIPcolor substrate solution.
We synthesized a polyclonal antibody against a conserved (identicalin mouse and rat and conserved in human) amino terminal peptide(S45-K65 in mKCNA10) of KCNA10. To assess the specificity ofthe antibody, we tested its ability to recognize in vitro translatedKCNA10 protein and distinguish it from Kv1.3 protein, the Kchannel protein most closely related to KCNA10 (17). Althoughanti-KCNA10 recognized a single band with an approximate molecularweight of 65 kd, which is nearly identical to that predictedby the primary sequence, it did not detect Kv1.3 protein (Figure 1a).Preimmune serum labeled neither KCNA10 nor Kv1.3 (Figure 1a).The specificity of the antibody was tested further in competitionstudies with the peptide used to generate anti-KCNA10. As shownin Figure 1b, the signal obtained with anti-KCNa10 was nearlycompletely abolished by the addition of the peptide. KCNA10is the only Kv channel gene that is more abundantly expressedin kidney than in brain (18). Figure 1c confirms that KCNA10protein is highly expressed in kidney and readily detectablein both crude and purified membrane vesicles preparations. Brushborder membranes, largely composed of apical membranes of proximaltubules, contain more KCNA10 protein than crude membranes, suggestingthat KCNA10 may be a proximal tubular K channel.
Figure 1. Specificity of a polyclonal antibody raised against KCNA10 protein. (A) Western blot showing that anti-KCNA10 labels in vitro translated hKCNA10 (approximately 65 kd) but not hKv1.3 (channel protein most closely related to KCNA10). Preimmune serum did not label any protein. (B) The anti-KCNA10 signal can be blocked by the antigenic peptide. Each lane contains 100 µg of crude rat renal membrane proteins separated by electrophoresis on a 10% SDS polyacrylamide. The lanes were cut out, and a Western blot was performed as described in Materials and Methods. The first lane (anti-KCNA10) was incubated with the primary antibody alone. The other lane (anti-KCNA10+ peptide) was incubated with anti-KCNA10 serum, which had been preincubated with the antigenic peptide. The signal detected by anti-KCNA10 crude renal membranes is of the expected size and is blocked by the antigenic peptide. (C) KCNA10 protein co-purifies with rat renal brush border membranes. Each lane contains 100 µg protein, and Western blotting is performed with the indicated primary antibody. KCNA10 (65-kd band) is relatively more abundant in brush border membranes than in crude membranes. The identity of the higher band is unclear and may represent KCNA10 protein complexes.
The cellular localization of KCNA10 in rat kidney was determinedby immunofluorescence. Anti-KCNA10 brightly labeled the apicalmembrane of proximal tubules, and a weaker signal was also evidentat the glomerulus (Figure 2, a through c). The distal tubuleand the limbs of Henle did not contain visible signals. Signalspecificity was ascertained in competition studies (Figure 2d)and by using preimmune serum (Figure 2e). No labeling was detectedin either study, confirming the specificity of the immunofluorescencesignal observed with anti-KCNA10. These data corroborate thefindings noted on Western Blot and indicate that KCNA10 is predominantlyexpressed at the apical membrane of the proximal tubule andat the glomerulus.
Figure 2. KCNA10 protein resides at the glomerulus and at the apical membrane of rat proximal tubular cells. (A) Renal expression KCNA10 protein expression was assessed by immunofluorescence. Anti-KCNA10 labeled only the glomerulus (*) and the apical membrane of proximal tubules (curved arrow). (B) Apical membrane labeling of proximal tubular cell. (C) Antigenic peptide blocks labeling by anti-KCNA10, indicating specificity of the signal. (D) Preimmune serum generates no signal. Magnifications: x10 in A; x20 in B, D, and E; x40 in C.
Although anti-KCNA10 detected hKCNA10 protein in Western blots(Figure 1, a and b), it was not useful for immunocytochemicalstudies, perhaps because the antigenic peptide is derived fromthe mouse sequence and only has 75% amino acid identity withhuman. Therefore, in situ hybridization was used to determinewhether KCNA10 expression in rat paralleled that in human. Theanti-sense probe strongly labeled proximal tubular cells (Figure 3, a and b).A positive signal was also clearly evident in glomerularendothelial cells (Figure 3 a and c) and in intrarenal arteries(Figure 3d). Signal specificity is illustrated in Figures 3e and 3f,where the sense probe failed to generate a detectablesignal. The in situ data are in agreement with those obtainedby immunofluorescence and document KCNA10 expression in proximaltubular cells and in glomerular endothelial cells. To determineif KCNA10 was expressed in other blood vessels, we evaluatedhuman cerebral artery by in situ hybridization. A positive signalwas detected in both endothelial and vascular smooth musclecells (Figure 4, a and b). Signal specificity was demonstratedusing the sense probe (Figure 4c).
Figure 3. KCNA10 gene expression detected by in situ hybridization in human glomerular endothelilium (*) and proximal tubular cells (curved arrow). (A) Anti-sense probe. (B and C) Anti-sense probe. (D) Anti-sense probe labels endothelium (curved arrow) and smooth muscle cells (*) of intrarenal artery. (E) No signal detected in tubules and glomerulus with sense probe. (F) No signal detected renal vessels with sense probe. Magnifications: x20 in A and E; x40 in B, C, D, and F.
Figure 4. KCNA10 expression in human vascular endothelilium (curved arrow) and vascular smooth muscle cells (*). (A) In situ hybridization with antisense probe. (B) Antisense probe. (C) Sense probe detected no signal. Magnifications: x10 in A; x40 in B and C.
In summary, these data indicate that anti-KCNA10 specificallyrecognizes KCNA10 protein, which is expressed at the apicalmembrane of rat proximal tubule. In situ hybridization studiesalso demonstrate that KCNA10 expression in rat kidney is similarto that in human.
Role of KCNA10 in Renal Proximal Tubule
At first glance, the high level of expression of KCNA10 proteinin proximal tubular cells, which are nonexcitable, is somewhatsurprising. Indeed, the activation threshold of most Kv channelsis >-60 mV and open only when the cell membrane depolarizesbeyond -60 mV. Such channels would remain closed in many epithelialcells with resting membrane potential more negative than -60mV under physiologic conditions. The proximal tubule containsseveral electrogenic transporters whose activities result insignificant cell membrane voltage depolarization. Sodium entryis facilitated by a favorable electrical chemical gradient,and large changes in membrane potential have been recorded duringsodium-coupled glucose and amino acid uptake. Proximal tubulecells have a resting membrane potential between -55 and -70mV but also depolarize by 10 to 15 mV when electrogenic transportprocesses such as Na-glucose or Na-amino acid transport areactivated (19). Depolarization of the cell membrane resultsin decreased in solute uptake; therefore, mechanisms that restorethe membrane potential are critical. Kv channels, such as KCNA10(activation treshold of -66 mV), are well suited for that taskbecause they allow K efflux and cell membrane hyperpolarizationwhen activated by membrane depolarization.
Kv channel activity has been detected at the apical membraneof proximal tubular cell using planar lipid bilayers (20). Inpatch clamp studies, the most commonly reported Kv channel belongsto the Maxi K class (21,22). In contrast, data obtained fromNorthern blotting, in situ hybridization, and immunocytochemistryclearly indicate the presence of at least two other types ofvoltage-gated potassium channels, namely KCNQ1 (23,24) and KCNA10.KCNQ1 forms a small conductance (2 to 10 pS) Kv channel andis thought to exist in vivo as a heteromultimeric complex withMInK, a small protein with a single transmembrane segment thatis highly expressed at the apical membrane of the proximal tubule.Recent data indicate that KCNQ1 may play an important role inthe regulation of sodium-coupled glucose and amino acid uptake,because mice deficient in MinK exhibit a reduction in glucoseand amino acid uptake by the proximal tubule (25). These dataconfirmed the hypothesis put forward by Sugimoto et al. (26).The current work indicates that KCNA10 is also present at theapical membrane of the proximal tubule. By analogy with KCNQ1,we postulate KCNA10 may participate in stabilizing membranevoltage during sodium entry at the apical membrane (Figure 5).
Figure 5. Proposed physiologic role of KCNA10 in the proximal tubule. Sodium entry depolarizes the cell membrane and K exit through KvLQT (Kv channel), KCNA10 and Kca (voltage and calcium gated K channel) at the apical and Kir 6.1 (inward rectifier) at the basolateral membrane repolarize the cell and maintain a favorable electrochemical gradient for Na, glucose, and amino acids absorption. MinK, accessory subunit of KvLQT1.
Role of KCNA10 in Glomerulus and Blood Vessels
Several types of K channels play a crucial role in the regulationof vascular smooth muscle contraction and, therefore, peripheralvascular resistance and BP (27,28). It appears that sustainedarterial contraction is critically dependent on the interplaybetween transport pathways that mediate Ca2+ entry and extrusion.Ca2+ entry is primarily mediated by voltage-dependent Ca2+ channelsand is therefore, regulated by changes in membrane voltage (29).Membrane hyperpolarization is mediated, in part, by the activationof K channels. Vasodilators modulate K channel activity in twoways. The K channel openers such as minoxidil, pinacidil, anddiazoxide activate K channels by binding to the channel protein(30). Others, such as nitric oxide, nitroglycerin, nitroprusside,and isoproterenol, are thought to increase intracellular cGMP,which in turn activates K channels. KCNA10 is expressed in bothendothelial (EC) and smooth muscle (SMC) cells, and its activityis regulated by cyclic nucleotides; therefore, it could participatein membrane repolarization and hyperpolarization.
In summary, KCNA10 is a cyclic nucleotide-gated Kv channel,expressed at the apical membrane of renal proximal tubular cells,well suited to stabilize cell membrane voltage during sustainedNa entry. The channel is also present in glomerular and vascularendothelial cells and in vascular smooth muscle cells. We postulatethat it regulates the membrane potential of these cells andcontributes to the process of vasodilatation.
Acknowledgments
This study was supported by the Veteran Administration MeritReview award to Gary V. Desir and by NIH grant DK48105B.
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Received for publication June 20, 2002.
Accepted for publication August 10, 2002.
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Abstract
Introduction
subunit of KCNA10 (
