Functional analysis and tissue-specific expression of Drosophila Na+,K+-ATPase subunits

We have previously purified and characterized a nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana [nerve antigen (NRV)] and identified two separate genes coding for three different proteins. All three proteins share homology with the beta subunits of Na+,K+-ATPase from various other species. In this study we have isolated a new Drosophila Na+,K+-ATPase alpha subunit cDNA clone (PSalpha; GenBank accession no. AF044974) and demonstrate expression of functional Na+,K+-ATPase activity when PSalpha mRNA is coinjected into Xenopus oocytes along with any of the three different Nrv mRNAs. Western blotting, RNase protection assays, and immunocytochemical staining of adult fly sections indicate that NRV2 is expressed primarily in the nervous system. Staining is most intense in the brain and thoracic ganglia and is most likely associated with neuronal elements. NRV1 is more broadly expressed in muscle and excretory tissue and also shows diffuse distribution in the nervous system. Similar to other species, Drosophila expresses multiple isoforms of Na+,K+-ATPase subunits in a tissue- and cell type-specific pattern. It will now be possible to use the advantages of Drosophila molecular and classical genetics to investigate the phenotypic consequences of altering Na+,K+-ATPase expression in various cell and tissue types.     

A putative fourth Na+,K(+)-ATPase alpha-subunit gene is expressed in testis

The Na+,K(+)-ATPase alpha subunit has three known isoforms, alpha 1, alpha 2 and alpha 3, each encoded by a separate gene. This study was undertaken to determine the functional status of a fourth human alpha-like gene, ATP1AL2. Partial genomic sequence analysis revealed regions exhibiting sequence similarity with exons 3-6 of the Na+,K(+)-ATPase alpha isoform genes. ATP1AL2 cDNAs spanning the coding sequence of a novel P-type ATPase alpha subunit were isolated from a rat testis library. The predicted polypeptide is 1028 amino acids long and exhibits 76-78% identity with the rat Na+,K(+)-ATPase alpha 1, alpha 2 and alpha 3 isoforms, indicating that ATP1AL2 may encode a fourth Na+,K(+)-ATPase alpha isoform. A 3.9-kb mRNA is expressed abundantly in human and rat testis.     

Distribution and elimination of RGH-5002 in rats

In this study we analysed the changes in the properties of rat cerebral cortex Na+K(+)-ATPase in streptozotocin induced diabetes (STZ-diabetes). Special attempt was made to determine whether insulin treatment of diabetic animals could restore the altered parameters of this enzyme. Na+/K(+)-ATPase activity was found to be decreased by 15% after 2 weeks, and by 37% after 4 weeks in diabetic rat brains with a parallel decrease in maximal capacity of low affinity ouabain binding sites. There was no significant change in the high affinity ouabain binding sites. The Kd values did not change significantly. Western blot analysis of brain Na+/K(+)-ATPase isoforms indicated a 61 +/- 5.8% and 20 +/- 2.8% decrease of the alpha 1 and alpha 3 isoforms, respectively in 4 weeks diabetic animals. Change in the amount of the alpha 2 isoform proved to be less characteristic. Both types of beta subunit isoform showed a significant decrease in four weeks diabetic rats. Our data indicate a good correlation in diabetic rats between changes in Na-/K(+)-ATPase activity, low affinity ouabain binding capacity and the level of alpha 1 isoform. While insulin treatment of diabetic animals restored the blood glucose level to normal, a complete reversal of diabetes induced changes in Na+/K(+)-ATPase activity, ouabain binding capacity and Na+/K(+)-ATPase isoform composition could not be achieved.     

Inhibition of rat Na+/K+-ATPase isoforms by endogenous digitalis extracts from neonatal human plasma

An unique endogenous digitalis-like factor (EDLF) has been previously purified from human newborn cord plasma and its differential effects tested on the three well defined functional isoforms (alpha1, alpha2 and alpha3) of the alpha subunits of Na+/K+-ATPase in rat. EDLF specifically inhibits the enzymatic activity. It differs from ouabain by three criteria: a preincubation with the membranes is required for full activity, no effect on the rat cerebral alpha3 isoform and a steep dose-response curve with the same apparent potency for rat alpha2 and alpha1 isoforms of high (10(-7) M) and low affinity (3 x 10(-5) M) for ouabain. These results indicate that the Na+/K+-ATPase inhibitor involved in the regulation of sodium and body fluid volume and present in neonate and adult human plasmas is distinct from ouabain.     

The influence of beta subunit structure on the stability of Na+/K(+)-ATPase complexes and interaction with K+

Heterologous expression of the beta subunit of H+/K(+)-ATPase (HK beta) with alpha subunits of Na+/K(+)-ATPase (NK alpha) in yeast leads to the formation of ouabain binding complexes, indicating assembly of the two subunits into active ion pumps (Eakle, K. A., Kim, K. S., Kabalin, M. A., and Farley, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2834-2838). Complexes of NK alpha and HK beta are less sensitive to inhibition of ouabain binding by K+, suggesting that HK beta lowers the affinity of K+ binding sites. This effect is particularly pronounced when HK beta is combined with the alpha 3 isoform of NK alpha. In this case, titration with K+ yields a biphasic curve, suggesting that there are two nonequivalent sites for K+ binding. Attempts at purifying complexes formed with either alpha 1 + HK beta or alpha 3 + HK beta using SDS extraction of microsomal membranes resulted in the loss of ouabain binding. Controls show that alpha 1 + beta 1 and alpha 3 + beta 1 complexes still retain ouabain binding after SDS extraction under the same conditions. This suggests that the HK beta subunit forms a less stable complex with NK alpha subunits. We have created chimeric beta subunits comprised of the amino-terminal cytoplasmic and transmembrane regions of HK beta combined with the carboxyl-terminal extracellular region of Na+/K(+)-ATPase beta 1 (HN beta 1) and the complementary chimera with amino-terminal cytoplasmic and transmembrane regions of beta 1 combined with the carboxyl-terminal extracellular region of HK beta (NH beta 1). When NH beta 1 is combined with either alpha 1 or alpha 3, the complexes show profiles of K+ inhibition of ouabain binding that are very similar to HK beta combined with either alpha 1 or alpha 3. The data suggest that the extracellular region of HK beta is primarily responsible for the effect on apparent K+ affinity. When the HN beta 1 subunit is expressed with the alpha 3 subunit, less than 5% of the amount of ouabain binding complexes are formed compared with HN beta 1 + alpha 1. This observation suggests that the HN beta 1 subunit either assembles poorly or forms an unstable complex with alpha 3. After SDS extraction, complexes of alpha 1 + NH beta 1 and alpha 3 + NH beta 1 retain ouabain binding, while alpha 1 + HN beta 1 complexes are sensitive to SDS extraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blood-brain barrier mechanisms involved in brain calcium and potassium homeostasis

This study examined the potential roles of the plasma membrane Ca2+-ATPase (PMCA) at the blood-CSF and blood-brain barriers in brain Ca2+ homeostasis and blood-brain barrier Na+/K+-ATPase subunits in brain K+ homeostasis. During dietary-induced hypo- and hypercalcemia (0.59+/-0.06 and 1.58+/-0.12 mM [Ca2+]) there was no significant change in choroid plexus PMCA (Western Blots) compared to normocalcemic rats (plasma [Ca2+]: 1.06+/-0.11 mM). In contrast, PMCA in cerebral microvessels isolated from hypocalcemic rats was 150% greater than that in controls (p<0.001). Comparison of the alpha3 subunit of Na+/K+-ATPase from cerebral microvessels isolated from hypo-, normo- and hyperkalemic rats (2.3+/-0.1, 3.9+/-0.1 and 7. 2+/-0.6 mM [K+]) showed a 75% reduction in the amount of this isoform during hyperkalemia. None of the other Na+/K+-ATPase isoforms varied with plasma [K+]. These results suggest that both PMCA and the alpha3 subunit of Na+/K+-ATPase at the blood-brain barrier play a role in maintaining a constant brain microenvironment during fluctuations in plasma composition.     

The alpha-subunit of the colonic H+,K+-ATPase assembles with beta1-Na+,K+-ATPase in kidney and distal colon

Previous experiments from our laboratory (Codina, J., Kone, B. C., Delmas-Mata, J. T., and DuBose, T. D., Jr. (1996) J. Biol. Chem. 271, 29759-29763) demonstrated that the alpha-subunit of the colonic H+, K+-ATPase (HKalpha2) requires coexpression with a beta-subunit to support H+/K+ transport in a heterologous expression system (Xenopus laevis oocytes). In these studies, HKalpha2 formed stable and functional alpha.beta complexes when coexpressed with either the rat beta1-subunit of the Na+,K+-ATPase or the beta-subunit of the gastric H+,K+-ATPase, suggesting that different beta-subunits may interact with HKalpha2. The present studies tested this hypothesis by development and application of a specific antibody against HKalpha2 peptide. Subsequently, immunoprecipitation experiments were performed to determine if HKalpha2 co-precipitates with the same beta-subunit in organs known to express HKalpha2 protein. The data demonstrate that HKalpha2 assembles with beta1-Na+,K+-ATPase in the renal medulla and in distal colon.     

The role of sports training in the efficiency of oxygen regimens in children in the North]

The comparative study of the sensitivity of Na+, K(+)-ATPase isozymes from cerebral cortex to ascorbate-dependent membrane peroxidation was conducted. With highly inactivated Na+, K(+)-ATPase the degree of inactivation of the SH-dependent ouabain-sensitive forms alpha+ (alpha 2 and alpha 3) is higher than glycoside-resistant isoform alpha 1. The process is accompanied by simultaneous lipid peroxidation and decrease of SH-groups amount in enzyme preparations. The combined nature of the oxidative Na+, K(+)-ATPase inactivation, accompanied by the direct oxidation of enzyme SH-groups and modification of lipid environment is supposed.     

Early prognostic biochemical indicators of fulminant hepatic failure

This article reviews related studies from the authors' laboratory, which focus on the regulation of vascular Na+,K+-ATPase in hypertension. Earlier studies, including the authors', suggested that Na-pump activity in cardiovascular tissues is subject to regulation during hypertension; most of these studies report a stimulation of the vascular enzyme during established stages of hypertension. To test hypothesis that in vascular smooth muscle, strain resulting from elevated pressure may be a signal initiating a cascade of events leading to increased expression of Na+,K+-ATPase, the authors used cell culture and the Flexercell Strain Unit to apply cyclical stretch to rat aortic smooth muscle cells (ASMC) for several days. These studies demonstrated that mechanical strain induces the upregulation of both the alpha-1 and alpha-2 subunits of Na+,K+-ATPase. Mechanisms underlying these changes appear to involve a transient increase in intracellular sodium entering the cell through stretch-activated channels. Calcium entering the cell via L-type channels did not affect stretch-induced upregulation of the alpha isoforms. In addition, protein kinase C inhibition resulted in inhibition of the Na-pump during stretch, but not under nonstretch conditions. The authors conclude that the stretch component of vascular pressure upregulates the Na+,K+-ATPase catalytic subunits. Intracellular sodium may be a signal for this regulation. In addition, phosphorylation by PKC may be important in stretch-induced short-term regulation of the vascular Na-pump.     

Glutamate uptake stimulates Na+,K+-ATPase activity in astrocytes via activation of a distinct subunit highly sensitive to ouabain

The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na+-dependent transporter. Evidence had been provided that Na+,K+-ATPase might be involved in this process. We have now measured the activity of Na+,K+-ATPase in cultured astrocytes, using ouabain-sensitive 86Rb uptake as an index. L-Glutamate increases glial Na+,K+-ATPase activity in a concentration-dependent manner with an EC50 = 67 microM. Both L- and D-aspartate, but not D-glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na+,K+-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain (K0.5 = 113 microM), compatible with the presence of an alpha1 isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain (K0.5 = 20 nM), thus revealing a high-affinity site akin to the alpha2 isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na+,K+-ATPase that can be mobilized in response to increased neuronal activity.     

Importance of chronopharmacokinetics in design and evaluation of transdermal drug delivery systems

BACKGROUND: Sodium-potassium-adenosinetriphosphatase (Na,K-ATPase) is the primary membrane enzyme responsible for the reabsorption of sodium ions in the kidney. It is known that in the nephron the major subunit isoforms of Na,K-ATPase are alpha 1 and beta 1. Previous reports on the presence of alpha 2 and alpha 3 isoforms in the kidney were mixed and controversial. METHODS: Techniques of ultrathin cryosectioning and immunoelectron microscopy were used to study the distribution of alpha subunit isoforms (alpha 1, alpha 2, alpha 3) and beta subunit (beta 1 isoform) of Na,K-ATPase in renal tubular cells. Western blot analysis was used to show the presence of the alpha 3 isoform in the extract of kidney mitochondria. RESULTS: We were able to confirm the previous finding that the alpha 1 isoform and the beta 1 isoform were the preponderant isoforms of the alpha and beta subunits of Na,K-ATPase in the basolateral membrane. In addition, we unexpectedly found the presence of the alpha 3 isoform in the mitochondria of rat renal tubular cells. The alpha 2 and alpha 3 isoforms were not observed in either the apical or basolateral membrane. CONCLUSIONS: Both immunoelectron microscopy and Western blot analysis of the rat kidney mitochondria confirm the presence of the alpha 3 isoform of Na,K-ATPase in the rat kidney mitochondria. The function of this enzyme in the mitochondria is not clear at this time.     

Neurotoxic effects of sodium nitroprusside in rat hippocampal slices

The effect of a permanent transection on myelin gene expression in a regenerating sciatic nerve and in an adult sciatic nerve was compared to establish the degree of axonal control exerted upon Schwann cells in each population. First, the adult sciatic nerve was crushed, and the distal segment allowed to regenerate. At 12 days post-crush, the sciatic nerve was transected distal to the site of crush to disrupt the Schwann cell-axonal contacts that had reformed. Messenger RNA (mRNA) levels coding for five myelin proteins were assayed in the distal segment of the crush-transected nerve after 9 days and were compared to corresponding levels in the distal segments of sciatic nerves at 21 days post-crush and 21 days post-transection using Northern blot and slot-blot analysis. Levels of mRNAs found in the distal segment of the transected and crush-transected nerve suggested that Schwann cells in the regenerating nerve and in the mature adult nerve are equally responsive to axonal influences. The crush-transected model allowed the genes that were studied to be classified according to their response to Schwann cell-axonal contact. The levels of mRNAs were 1) down-regulated to basal levels (P0 and MBP mRNAs), 2) down-regulated to undetectable levels (myelin-associated glycoprotein mRNAs), 3) upregulated (mRNAs encoding 2'3'-cyclic nucleotide phosphodiesterase and beta-actin), or 4) not stringently controlled by the removal of Schwann cell-axonal contact (proteolipid protein mRNAs). This novel experimental model has thus provided evidence that the expression of some of the important myelin genes during peripheral nerve regeneration is dependent on continuous signals from the ingrowing axons.     

Dopamine-induced endocytosis of Na+,K+-ATPase is initiated by phosphorylation of Ser-18 in the rat alpha subunit and Is responsible for the decreased activity in epithelial cells

Dopamine inhibits Na+,K+-ATPase activity in renal tubule cells. This inhibition is associated with phosphorylation and internalization of the alpha subunit, both events being protein kinase C-dependent. Studies of purified preparations, fusion proteins with site-directed mutagenesis, and heterologous expression systems have identified two major protein kinase C phosphorylation residues (Ser-11 and Ser-18) in the rat alpha1 subunit isoform. To identify the phosphorylation site(s) that mediates endocytosis of the subunit in response to dopamine, we have performed site-directed mutagenesis of these residues in the rat alpha1 subunit and expressed the mutated forms in a renal epithelial cell line. Dopamine inhibited Na+,K+-ATPase activity and increased alpha subunit phosphorylation and clathrin-dependent endocytosis into endosomes in cells expressing the wild type alpha1 subunit or the S11A alpha1 mutant, and both effects were blocked by protein kinase C inhibition. In contrast, dopamine did not elicit any of these effects in cells expressing the S18A alpha1 mutant. While Ser-18 phosphorylation is necessary for endocytosis, it does not affect per se the enzymatic activity: preventing endocytosis with wortmannin or LY294009 blocked the inhibitory effect of dopamine on Na+,K+-ATPase activity, although it did not alter the increased alpha subunit phosphorylation induced by this agonist. We conclude that dopamine-induced inhibition of Na+, K+-ATPase activity in rat renal tubule cells requires endocytosis of the alpha subunit into defined intracellular compartments and that phosphorylation of Ser-18 is essential for this process.     

Alpha 2 mRNA of Na+K+ ATPase is increased in astroctyes of rat hippocampus after treatment with kainic acid

The Na+K+ ATPase (Na+ pump) plays a central role in regulating cation homeostasis and is thought to have an important role in cell proliferation. The multitude of subunit isoforms comprising the functional Na+K+ ATPase has raised the possibility that specific subunit isoform combinations may be involved in different cellular processes. We have investigated the involvement of the specific isoforms in neurons and glia at the site of a CNS lesion. Intracerebroventricular injection of kainic acid was used to induce neuronal cell loss and reactive gliosis in rat hippocampus and levels of Na+K+ ATPase subunit isoform mRNA levels were determined in cells of rat hippocampus using in situ hybridization. alpha 2 mRNA levels increased 35-40% in CA1 and CA3 astrocytes between 1-3 weeks after KA injection with no significant change in other subunit isoform mRNA levels. In addition alpha 3 mRNA levels in CA1 pyramidal neurons were decreased by approx. 35%. Small neurons in the CA1 and CA3 region showed no changes in mRNA levels for any of the Na+K+ ATPase subunit isoforms. These results may indicate a possible role for alpha 2 subunit isoform in the conversion of glial cells from a normal phenotype to the reactive phenotype characteristic in this model of CNS injury.     

Photoinactivation of fluorescein isothiocyanate-modified Na,K-ATPase by 2'(3')-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5'-diphosphate. Abolition of E1 and E2 partial reactions by sequential block of high and low affinity nucleotide sites

The Na,K-ATPase activity of the sodium pump exhibits apparent multisite kinetics toward ATP, a feature that is inherent to the minimal enzyme unit, the alpha beta protomer. We have argued that this should arise from separate catalytic and noncatalytic sites on the alpha beta protomer as fluorescein isothiocyanate (FITC) blocks a high affinity ATP site on all alpha subunits and yet the modified Na, K-ATPase retains a low affinity response to nucleotides (Ward, D. G., and Cavieres, J. D. (1996) J. Biol. Chem. 271, 12317-12321). We now find that 2'(3')-O-(2,4,6-trinitrophenyl)8-azido-adenosine 5'-diphosphate (TNP-8N3-ADP), a high affinity photoactivatable analogue of ATP, can inhibit the K+-phosphatase activity of the FITC-modified enzyme during assays in dimmed light. The inhibition occurs with a Ki of 140 microM at 20 mM K+; it requires the adenine ring as 2'(3')-O-(2,4 6-trinitrophenyl) (TNP)-UDP or TNP-uridine are less potent and 2,4,6-trinitrobenzene-sulfonate is ineffective. Under irradiation with UV light, TNP-8N3-ADP inactivates the K+-phosphatase activity of the fluorescein-enzyme and also its phosphorylation by [32P]Pi. The photoinactivation process is stimulated by Na+ or Mg2+, and is inhibited by K+ or excess TNP-ADP. In the presence of 50 mM Na+ and 1 mM Mg2+, TNP-8N3-ADP photoinactivates with a K0.5 of 15 microM. Furthermore, TNP-8N3-ADP photoinactivates the FITC-modified, solubilized alpha beta protomers, even more effectively than the membrane-bound fluorescein-enzyme. These results strongly suggest that catalytic and allosteric ATP sites coexist on the alpha beta protomer of Na,K-ATPase.     

Structure-function study on gastric proton pump

H+, K(+)-ATPase is a proton pump responsible for gastric acid secretion. It actively transport proton and K+ coupled with the hydrolysis of ATP, resulting in the formulation of a 10(6) fold proton gradient across the plasma membrane of parietal cells. The pump belongs to a family of P-type ATPases which include the Na+ pump (Na+, K(+)-ATPase) and the Ca2+ pump (Ca(2+)-ATPase). This review focuses on the structure-function relationship of this proton pump by using functional antibodies, specific inhibitor(s), a fluorescent reagent and site-directed mutants. First we prepared monoclonal antibodies which modified the functions of the H+, K(+)-ATPase . One of the antibodies, HK2032 inhibited the H+, K(+)-ATPase activity and the chloride conductance in gastric vesicles opened by S-S cross-linking, suggesting that the chloride pathway is in the H+, K(+)-ATPase molecule, and that the H+, K(+)-ATPase is a multi-functional molecule. Other antibody, HK4001 inhibited the H+, K(+)-ATPase activity by inhibiting its phosphorylation step. By using this antibody we found an H+, K(+)-ATPase isoform in the rabbit distal colon. Second we found that scopadulcic acid B, a main ingredient of Paraguayan traditional herb, is an inhibitor specific for the H+, K(+)-ATPase. This compound inhibited the H+, K(+)-ATPase activity by stabilizing the K(+)-form of the enzyme. Third we studied the conformational changes of the H+, K(+)-ATPase by observing the fluorescence of FITC-labeled enzyme. H+, K(+)-ATPase did not utilize acetylphosphate instead the ATP as an energy source of active transport, suggesting that the energy transduction system is not common among P-type ATPases. Finally we constructed a functional expression system of the H+, K(+)-ATPase in human kidney cells. By using this functional expression system in combination with site-directed mutagenesis, we studied the significance of amino acid residues in the catalytic centers (a phosphorylation site and an ATP binding site) and the putative cation binding sites. We newly found the sites determining the affinity for cations.     

Glucose decreases Na+,K+-ATPase activity in pancreatic beta-cells. An effect mediated via Ca2+-independent phospholipase A2 and protein kinase C-dependent phosphorylation of the alpha-subunit

In the pancreatic beta-cell, glucose-induced membrane depolarization promotes opening of voltage-gated L-type Ca2+ channels, an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), and exocytosis of insulin. Inhibition of Na+,K+-ATPase activity by ouabain leads to beta-cell membrane depolarization and Ca2+ influx. Because glucose-induced beta-cell membrane depolarization cannot be attributed solely to closure of ATP-regulated K+ channels, we investigated whether glucose regulates other transport proteins, such as the Na+,K+-ATPase. Glucose inhibited Na+,K+-ATPase activity in single pancreatic islets and intact beta-cells. This effect was reversible and required glucose metabolism. The inhibitory action of glucose was blocked by pretreatment of the islets with a selective inhibitor of a Ca2+-independent phospholipase A2. Arachidonic acid, the hydrolytic product of this phospholipase A2, also inhibited Na+, K+-ATPase activity. This effect, like that of glucose, was blocked by nordihydroguaiaretic acid, a selective inhibitor of the lipooxygenase metabolic pathway, but not by inhibitors of the cyclooxygenase or cytochrome P450-monooxygenase pathways. The lipooxygenase product 12(S)-HETE (12-S-hydroxyeicosatetranoic acid) inhibited Na+,K+-ATPase activity, and this effect, as well as that of glucose, was blocked by bisindolylmaleimide, a specific protein kinase C inhibitor. Moreover, glucose increased the state of alpha-subunit phosphorylation by a protein kinase C-dependent process. These results demonstrate that glucose inhibits Na+, K+-ATPase activity in beta-cells by activating a distinct intracellular signaling network. Inhibition of Na+,K+-ATPase activity may thus be part of the mechanisms whereby glucose promotes membrane depolarization, an increase in [Ca2+]i, and thereby insulin secretion in the pancreatic beta-cell.     

Heteromultimeric delayed-rectifier K+ channels in schwann cells: developmental expression and role in cell proliferation

Schwann cells (SCs) are responsible for myelination of nerve fibers in the peripheral nervous system. Voltage-dependent K+ currents, including inactivating A-type (KA), delayed-rectifier (KD), and inward-rectifier (KIR) K+ channels, constitute the main conductances found in SCs. Physiological studies have shown that KD channels may play an important role in SC proliferation and that they are downregulated in the soma as proliferation ceases and myelination proceeds. Recent studies have begun to address the molecular identity of K+ channels in SCs. Here, we show that a large repertoire of K+ channel alpha subunits of the Shaker (Kv1.1, Kv1.2, Kv1.4, and Kv1.5), Shab (Kv2.1), and Shaw (Kv3.1b and Kv3.2) families is expressed in mouse SCs and sciatic nerve. We characterized heteromultimeric channel complexes that consist of either Kv1.5 and Kv1.2 or Kv1.5 and Kv1.4. In postnatal day 4 (P4) sciatic nerve, most of the Kv1.2 channel subunits are involved in heteromultimeric association with Kv1.5. Despite the presence of Kv1. 1 and Kv1.2 alpha subunits, the K+ currents were unaffected by dendrotoxin I (DTX), suggesting that DTX-sensitive channel complexes do not account substantially for SC KD currents. SC proliferation was found to be potently blocked by quinidine or 4-aminopyridine but not by DTX. Consistent with previous physiological studies, our data show that there is a marked downregulation of all KD channel alpha subunits from P1-P4 to P40 in the sciatic nerve. Our results suggest that KD currents are accounted for by a complex combinatorial activity of distinct K+ channel complexes and confirm that KD channels are involved in SC proliferation.