Synthesis and biological evaluation of myoseverin derivatives: microtubule assembly inhibitors.

Myoseverin, a trisubstituted purine, inhibits microtubule assembly in vitro, interferes with normal mitotic spindle assembly, and arrests the cell cycle in mitosis in U937 cells. We synthesized a variety of myoseverin derivatives and screened them for inhibition of spindle assembly in Xenopus egg extracts and for microtubule disassembly in vitro. Selected compounds were tested against 60 cancer cell lines at the National Cancer Institute as possible anticancer drug candidates.

Mitotic spindle assembly in vitro.

The protocols in this unit describe the preparation of materials for an in vitro assay of mitotic spindle assembly in Xenopus egg extracts. Fluorochrome-labeled tubulin is used to visualize microtubule asters and spindles.

A cyclin-dependent kinase inhibitor inducing cancer cell differentiation: biochemical identification using Xenopus egg extracts.

Cellular differentiation is a complex process involving growth arrest, exit from the cell cycle, and expression of differentiated cell-type-specific functions. To identify small molecules promoting this process, a chemical library was screened by using a myeloid leukemic cell line that retained the potential to differentiate in culture. In the presence of a purine derivative, aminopurvalanol (AP), cells acquired phenotypic characteristics of differentiated macrophages and became arrested in the cell cycle with a 4N DNA content. AP also inhibited mitosis in Xenopus egg extracts, suggesting that it acted on an evolutionarily conserved cell cycle regulatory pathway. Affinity chromatography and biochemical reconstitution experiments with Xenopus egg extracts identified cyclin-dependent kinase (CDK) 1-cyclin B as a target of the compound. Although AP potently inhibited immunoprecipitates of both human CDK1 and CDK2 from human leukemic cell extracts, our results indicate that the compound preferentially targets the G2/M-phase transition in vivo.

Identification of cell type-specific promoter elements associated with the rat tyrosine hydroxylase gene using transgenic founder analysis.

Transcriptional regulatory elements capable of directing transgene expression to individual cells are powerful tools for manipulating a given CNS circuit. Delineating these elements via traditional transgenic analysis is both costly and labor intensive. Here we have used the rat tyrosine hydroxylase (TH) promoter as a model to describe and validate the use of founder animals for systematic promoter studies. No significant differences were found when data obtained from founder animals expressing a 6.0 kb TH promoter directing LacZ were compared with animals derived from an analogous transgenic line. Subsequent studies with founder animals expressing beta-galactosidase directed by various lengths of rat TH promoter revealed different patterns of expression. Specifically, a locus coeruleus regulatory domain was localized between 3.4 and 6.0 kb of the rat TH promoter, a hypothalamic regulatory domain between 2.5 and 3.4 kb and a brainstem regulatory domain between 0.8 and 6.0 kb. At least one element of a midbrain specific regulatory domain was within 2.5 kb of the transcriptional start site. Olfactory bulb specific elements however appeared to reside outside of the sequences tested. Specific patterns of ectopic gene expression were also observed suggesting the presence of negative regulatory elements. Thus, TH appears to be regulated in a complex modular fashion by both positive and negative regulatory elements. Taken together, this study demonstrates the feasibility and reliability of founder analysis for promoter studies of genes expressed in complex spatial and temporal patterns.

Deficient development and maintenance of postsynaptic specializations in mutant mice lacking an 'adult' acetylcholine receptor subunit.

At many synapses, 'fetal' neurotransmitter receptor subunits are replaced by 'adult' subunits as development proceeds. To assess the significance of such transitions, we deleted the gene encoding the adult acetylcholine receptor (AChR) epsilon subunit, which replaces its fetal counterpart, the gamma subunit, at the skeletal neuromuscular junction during early postnatal life. Several aspects of postnatal maturation, including synapse elimination, proceeded normally in the absence of the adult AChR, but structural development of the endplate was compromised. Later, inadequate compensation by the gamma subunit led to severely reduced AChR density in mutant endplates relative to controls. This decreased density led to a profound reorganization of AChR-associated components of the postsynaptic membrane and cytoskeleton. Together, these results suggest novel roles for AChRs in assembly of the postsynaptic apparatus.

Distal regulatory elements control MRF4 gene expression in early and late myogenic cell populations.

MRF4 is a muscle-specific transcription factor that belongs to a family of basic helix-loop-helix proteins known as the myogenic regulatory factors (MRFs). In vitro studies have shown that expression of the MRF4 gene is controlled by a proximal promoter element (-336 to +71) that binds the muscle-specific transcription factors MEF2 and myogenin to activate transcription. To examine further the regulatory elements necessary for endogenous MRF4 gene expression during development, transgenic mice were generated that contained either a proximal MRF4 promoter-LacZ reporter gene (-336 MRF4-nLacZ) or a MRF4-LacZ reporter gene containing 8.5 kb of 5' flanking sequence (-8500 MRF4-nLacZ). Characterization of individual transgenic mouse lines throughout development revealed that expression of both transgenes is restricted to skeletal muscle tissue. However, unlike previous in vitro data, the proximal promoter transgene exhibits only limited transcriptional activity at all developmental time points, whereas the -8500 MRF4-nLacZ lines fully recapitulate the later developmental expression patterns and exhibit transcription in myotomal cells during somitic differentiation. Tissue culture analysis of myogenic cells isolated from E12.5, E16.5, and adults confirmed that the -8500 MRF4-nLacZ transgene is expressed in greater than 90% of the myotubes for all myogenic populations. These results indicate that 8.5 kb of MRF4 5' flanking sequence contains all the regulatory elements necessary for late MRF4 expression and that at least some of these elements lie upstream of the -336 proximal promoter. It is also likely that distant upstream regulatory sequences control early somitic MRF4 expression. These findings, coupled with previous in vitro studies, suggest that the early and late developmental expression patterns of the MRF4 gene are controlled by distinct sets of regulatory elements.

Subtle neuromuscular defects in utrophin-deficient mice.

Utrophin is a large cytoskeletal protein that is homologous to dystrophin, the protein mutated in Duchenne and Becker muscular dystrophy. In skeletal muscle, dystrophin is broadly distributed along the sarcolemma whereas utrophin is concentrated at the neuromuscular junction. This differential localization, along with studies on cultured cells, led to the suggestion that utrophin is required for synaptic differentiation. In addition, utrophin is present in numerous nonmuscle cells, suggesting that it may have a more generalized role in the maintenance of cellular integrity. To test these hypotheses we generated and characterized utrophin-deficient mutant mice. These mutant mice were normal in appearance and behavior and showed no obvious defects in muscle or nonmuscle tissue. Detailed analysis, however, revealed that the density of acetylcholine receptors and the number of junctional folds were reduced at the neuromuscular junctions in utrophin-deficient skeletal muscle. Despite these subtle derangements, the overall structure of the mutant synapse was qualitatively normal, and the specialized characteristics of the dystrophin-associated protein complex were preserved at the mutant neuromuscular junction. These results point to a predominant role for other molecules in the differentiation and maintenance of the postsynaptic membrane.

Cloning and expression of full length mouse utrophin: the differential association of utrophin and dystrophin with AChR clusters.

We have cloned and sequenced mouse utrophin cDNA, and successfully expressed full length utrophin (400 kDa) in both muscle and non-muscle cells. The expression of recombinant utrophin is compared with that of its homologue, dystrophin (427 kDa). We demonstrate that recombinant utrophin is targeted into agrin-induced acetylcholine receptor (AChR) clusters, while recombinant dystrophin is evenly distributed along cell membranes in cultured Sol 8 muscle cells. This observation suggests that utrophin and dystrophin may interact with different cytoskeletal proteins. The C-terminal domains are found to be responsible for the association of utrophin with AChR clusters.

Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes.

In the experiments here, the developmental expression of the functional Ca(2+)-independent, depolarization-activated K+ channel currents, Ito and IK, and of the voltage-gated K+ channel (Kv) alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 in rat ventricular myocytes were examined quantitatively. Using the whole-cell patch clamp recording method, the properties and the densities of Ito and IK in ventricular myocytes isolated from postnatal day 5 (P5), 10 (P10), 15 (P15), 20 (P20), 25 (P25), 30 (P30), and adult (8-12 wk) rats were characterized and compared. These experiments revealed that mean Ito densities increase fourfold between birth and P30, whereas IK densities vary only slightly. Neither the time- nor the voltage-dependent properties of the currents vary measurably, suggesting that the subunits underlying functional Ito and IK channels are the same throughout postnatal development. In parallel experiments, the developmental expression of each of the voltage-gated K+ channel alpha subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, was examined quantitatively at the mRNA and protein levels using subunit-specific probes. RNase protection assays revealed that Kv1.4 message levels are high at birth, increase between P0 and P10, and subsequently decrease to very low levels in adult rat ventricles. The decrease in message is accompanied by a marked reduction in Kv1.4 protein, consistent with our previous suggestion that Kv1.4 does not contribute to the formation of functional K+ channels in adult rat ventricular myocytes. In contrast to Kv1.4, the mRNA levels of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 increase (three- to five-fold) between birth and adult. Western analyses, however, revealed that the expression patterns of these subunits proteins vary in distinct ways: Kv1.2 and Kv4.2, for example, increase between P5 and adult, whereas Kv1.5 remains constant and Kv2.1 decreases. Throughout development, therefore, there is a mismatch between the numbers of Kv alpha subunits expressed and the functional voltage-gated K+ channel currents distinguished electrophysiologically in rat ventricular myocytes. Alternative experimental approaches will be required to define directly the Kv alpha subunits that underlie functional voltage-gated K+ channels in these (and other) cells. In addition, the finding that Kv alpha subunit protein expression levels do not necessarily mirror mRNA levels suggests that caution should be exercised in attempting functional interpretations of observed changes in mRNA levels alone.

Maturation of the acetylcholine receptor in skeletal muscle: regulation of the AChR gamma-to-epsilon switch.

During the development of the mammalian neuromuscular junction, acetylcholine receptors (AChRs) become localized to the postsynaptic muscle membrane. As this process nears completion, the fetal form of the receptor, containing a gamma subunit (composition alpha 2 beta gamma delta) is gradually replaced by an epsilon subunit-containing adult form (alpha 2 beta epsilon delta). To understand how this transition is controlled, we compared the expression and regulation of the AChR gamma and epsilon subunits in developing, adult, and cultured muscles. Immunostaining with subunit-specific antibodies showed that replacement of gamma subunit- by epsilon subunit-containing AChRs occurs largely during the first postnatal week in fast-twitch muscles, and occurs homogeneously throughout individual endplates. In the slow-twitch soleus, however, this transition is delayed, and in the multiply innervated slow fibers of extraocular muscle, gamma subunit expression persists into adulthood. The transcriptional bases of the AChR subunit transition, and of these intermuscular variations, were demonstrated in mice bearing transgenes containing promoter elements from the AChR gamma and epsilon subunit genes, each coupled to a nuclear-localized beta-galactosidase (nlacZ) reporter. We show that transgene expression is stimulated by the nerve-derived inducer of AChR expression, ARIA, in myotubes cultured from gamma-nlacZ as well as epsilon-nlacZ mice. However, the expression of gamma-nlacZ, but not epsilon-nlacZ, is increased by treatment of myotubes with TTX, and the ARIA sensitivity of gamma-nlacZ is dependent on the electrical state of the myotube. Thus, the promoters of the gamma and epsilon subunit genes may integrate ARIA- and activity-dependent signals in different ways to generate their complementary patterns of expression.

Distinct regulatory elements control muscle-specific, fiber-type-selective, and axially graded expression of a myosin light-chain gene in transgenic mice.

The fast alkali myosin light chain 1f/3f (MLC1f/3f) gene is developmentally regulated, muscle specific, and preferentially expressed in fast-twitch fibers. A transgene containing an MLC1f promoter plus a downstream enhancer replicates this pattern of expression in transgenic mice. Unexpectedly, this transgene is also expressed in a striking (approximately 100-fold) rostrocaudal gradient in axial muscles (reviewed by J. R. Sanes, M. J. Donoghue, M. C. Wallace, and J. P. Merlie, Cold Spring Harbor Symp. Quant. Biol. 57:451-460, 1992). Here, we analyzed the expression of mutated transgenes to map sites necessary for muscle-specific, fiber-type-selective, and axially graded expression. We show that two E boxes (myogenic factor binding sites), a homeodomain (hox) protein binding site, and an MEF2 site, which are clustered in an approximately 170-bp core enhancer, are all necessary for maximal transgene activity in muscle but not for fiber-type- or position-dependent expression. A distinct region within the core enhancer promotes selective expression of the transgene in fast-twitch muscles. Sequences that flank the core enhancer are also necessary for high-level activity in transgenic mice but have little influence on activity in transfected cells, suggesting the presence of regions resembling matrix attachment sites. Truncations of the MLC1f promoter affected position-dependent expression of the transgene, revealing distinct regions that repress transgene activity in neck muscles and promote differential expression among intercostal muscles. Thus, the whole-body gradient of expression displayed by the complete transgene may reflect the integrated activities of discrete elements that regulate expression in subsets of muscles. Finally, we show that transgene activity is not significantly affected by deletion or overexpression of the myoD gene, suggesting that intermuscular differences in myogenic factor levels do not affect patterns of transgene expression. Together, our results provide evidence for at least nine distinct sites that exert major effects on the levels and patterns of MLC1f expression in adult muscles.

ARIA/HRG regulates AChR epsilon subunit gene expression at the neuromuscular synapse via activation of phosphatidylinositol 3-kinase and Ras/MAPK pathway.

AChR-inducing activity (ARIA)/heregulin, a ligand for erbB receptor tyrosine kinases (RTKs), is likely to be one nerve-supplied signal that induces expression of acetylcholine receptor (AChR) genes at the developing neuromuscular junction. Since some RTKs act through Ras and phosphatidylinositol 3-kinase (PI3K), we investigated the role of these pathways in ARIA signaling. Expression of activated Ras or Raf mimicked ARIA-induction of AChR epsilon subunit genes in muscle cells; whereas dominant negative Ras or Raf blocked the effect of ARIA. ARIA rapidly activated erk1 and erk2 and inhibition of both erks also abolished the effect of ARIA. ARIA stimulated association of PI3K with erbB3, expression of an activated PI3K led to ARIA-independent AChR epsilon subunit expression, and inhibition of PI3K abolished the action of ARIA. Thus, synaptic induction of AChR genes requires activation of both Ras/MAPK and PI3K signal transduction pathways.

Structural organization of the human and mouse laminin beta2 chain genes, and alternative splicing at the 5' end of the human transcript.

We have determined the structural organization of the human and mouse genes that encode the laminin beta2 chain (s-laminin), an essential component of the basement membranes of the neuromuscular synapse and the kidney glomerulus. The human and mouse genes have a nearly identical exon-intron organization and are the smallest laminin chain genes characterized to date, due to the unusually small size of their introns. The laminin beta2 chain genes of both species consist of 33 exons that span

Defective neuromuscular synaptogenesis in agrin-deficient mutant mice.

During neuromuscular synapse formation, motor axons induce clustering of acetylcholine receptors (AChRs) in the muscle fiber membrane. The protein agrin, originally isolated from the basal lamina of the synaptic cleft, is synthesized and secreted by motoneurons and triggers formation of AChR clusters on cultured myotubes. We show here postsynaptic AChR aggregates are markedly reduced in number, size, and density in muscles of agrin-deficient mutant mice. These results support the hypothesis that agrin is a critical organizer of postsynaptic differentiation does occur in the mutant, suggesting the existence of a second-nerve-derived synaptic organizing signal. In addition, we show that intramuscular nerve branching and presynaptic differentiation are abnormal in the mutant, phenotypes which may reflect either a distinct effect of agrin or impaired retrograde signaling from a defective postsynaptic apparatus.

The synapse-associated protein rapsyn regulates tyrosine phosphorylation of proteins colocalized at nicotinic acetylcholine receptor clusters.

Protein tyrosine phosphorylation has been suggested to play an important role in the clustering of the nicotinic acetylcholine receptor (AChR) at the developing neuromuscular junction. Recent studies have shown that the 43-kDa synapse-associated protein rapsyn induces clustering of the AChR in heterologous expression systems. In this study we examined whether tyrosine phosphorylation is involved in this rapsyn-induced AChR clustering. Rapsyn-induced AChR clusters in fibroblasts contain phosphotyrosine, as detected using immunofluorescent labeling with anti-phosphotyrosine antibodies. No anti-phosphotyrosine staining of rapsyn clusters is seen in the absence of AChR expression, indicating that the AChR is required for the appearance of phosphotyrosine at clusters. In addition, coexpression of rapsyn with the AChR induces the tyrosine phosphorylation of the beta amd delta subunits of the AChR. Surprisingly, mutation of the tyrosine phosphorylation sites in the AChR did not inhibit rapsyn-induced clustering of the AChR and clusters of the mutant AChRs still contained high levels of phosphotyrosine. Experiments with single AChR subunits demonstrate that the alpha subunit of the AChR appears to be necessary and sufficient for codistribution of phosphotyrosine with rapsyn-induced clusters of AChR subunits. Finally, transfection of cells with rapsyn activates cellular protein tyrosine kinase activity, resulting in the tyrosine phosphorylation of several membrane-associated proteins. These results suggest that rapsyn may therefore regulate clustering at least in part by regulating the tyrosine phosphorylation of cellular proteins.

The Eph kinase ligand AL-1 is expressed by rostral muscles and inhibits outgrowth from caudal neurons.

In the peripheral nervous system, neurons derived from specific rostrocaudal levels of the neuraxis selectively synapse on targets that arise from corresponding body positions. To identify molecules involved in such position-dependent connectivity, we used subtractive hybridization to isolate genes selectively expressed in rostral or caudal skeletal muscle. One mRNA that was more abundant in neck than in hindlimb muscles encoded the mouse ortholog of human AL-1 and chick RAGS, membrane-associated ligands of Eph tyrosine kinases that have recently been implicated in cortical axon fasciculation and retinotectal connectivity, respectively. We show here that mouse AL-1 is expressed in discrete regions of the central and peripheral nervous systems and in a subset of developing skeletal muscles. The abundance of AL-1 RNA in immortalized myogenic cell lines derived from rostral muscles is higher than in caudally derived lines, suggesting that levels are heritably maintained. Growth of neurites from cultured sensory ganglia and spinal cords is specifically inhibited by cells expressing AL-1, suggesting that this molecule could serve to guide peripheral axons. The inhibitory effects of AL-1 are position dependent, such that axons derived from caudal (lumbar) ganglia are more affected than those derived from rostral (cervical) ganglia. Together, these results support the notion that Eph kinases and their ligands regulate topographically appropriate neural connectivity in the peripheral nervous system, as well as in the central nervous system.

Overexpression of myogenin in muscles of transgenic mice: interaction with Id-1, negative crossregulation of myogenic factors, and induction of extrasynaptic acetylcholine receptor expression.

To investigate the role of myogenin in regulating acetylcholine receptor expression in adult muscle, this muscle-specific basic helix-loop-helix transcription factor was overexpressed in transgenic mice by using regulatory elements conferring strong expression confined to differentiated postmitotic muscle fibers. Many of the transgenic mice died during the first postnatal week, but those that survived into adulthood displayed normal muscle histology, gross morphology, and motor behavior. The mRNA levels of all five acetylcholine receptor subunits (alpha, beta, gamma, delta, and epsilon) were, however, elevated. Also, the level of receptor protein was increased and high levels of receptors were present throughout the extrasynaptic surface membrane of the muscle fibers. Thus, elevated levels of myogenin are apparently sufficient to induce acetylcholine supersensitivity in normally innervated muscle of adult mice. The high neonatal mortality rate of the mice overexpressing myogenin hindered the propagation of a stable line. In an attempt to increase survival, myogenin overexpressers were mated with a line of transgenic mice overexpressing Id-1, a negative regulator that interacts with the basic helix-loop-helix family of transcription factors. The Id-1 transgene apparently worked as a second site suppressor and abolished the high rate of neonatal mortality. This effect indicates that Id-1 and myogenin interact directly or indirectly in these animals. Further study indicated that myogenin overexpression had no effect on the level of endogenous myogenin mRNA, while the levels of myoD and MRF4 mRNAs were reduced. Overexpression of the negative regulator Id-1 increased the mRNA levels of all the myogenic factors. These findings are consistent with a hypothesis suggesting that myogenic factors are influenced by mechanisms that maintain cellular homeostasis.

Synapse-associated expression of an acetylcholine receptor-inducing protein, ARIA/heregulin, and its putative receptors, ErbB2 and ErbB3, in developing mammalian muscle.

Developing motor axons induce synaptic specializations in muscle fibers, including preferential transcription of acetylcholine receptor (AChR) subunit genes by subsynaptic nuclei. One candidate nerve-derived signaling molecule is AChR-inducing activity (ARIA)/heregulin, a ligand of the erbB family of receptor tyrosine kinases. Here, we asked whether ARIA and erbB kinases are expressed in patterns compatible with their proposed signaling roles. In developing muscle, ARIA was present not only at synaptic sites, but also in extrasynaptic regions of the muscle fiber. ARIA was synthesized, rather than merely taken up, by muscle cells, as indicated by the presence of ARIA mRNA in muscle and of ARIA protein in a clonal muscle cell line. ARIA-responsive myotubes expressed both erbB2 and erbB3, but little EGFR/erbB1 or erbB4. In adults, erbB2 and erbB3 were localized to the postsynaptic membrane. ErbB3 was restricted to the postsynaptic membrane perinatally, at a time when ARIA was still broadly distributed. Thus, our data are consistent with a model in which ARIA interacts with erbB kinases on the muscle cell surface to provide a local signal that induces synaptic expression of AChR genes. However, much of the ARIA is produced by muscle, not nerve, and the spatially restricted response may result from the localization of erbB kinases as well as of ARIA. Finally, we show that erbB3 is not concentrated at synaptic sites in mutant mice that lack rapsyn, a cytoskeletal protein required for AChR clustering, suggesting that pathways for synaptic AChR expression and clustering interact.

Failure of postsynaptic specialization to develop at neuromuscular junctions of rapsyn-deficient mice.

Of numerous synaptic components that have been identified, perhaps the best-studied are the nicotinic acetylcholine receptors (AChRs) of the vertebrate neuromuscular junction. AChRs are diffusely distributed on embryonic myotubes, but become highly concentrated (approximately 10,000 microns-2) in the postsynaptic membrane as development proceeds. At least two distinct processes contribute to this accumulation. One is local synthesis: subsynaptic muscle nuclei transcribe AChR subunit genes at higher rates than extra-synaptic nuclei, so AChR messenger RNA is concentrated near synaptic sites. Second, once AChRs have been inserted in the membrane, they form high-density clusters by tethering to a subsynaptic cytoskeletal complex. A key component of this complex is rapsyn, a peripheral membrane protein of relative molecular mass 43K (refs 4, 5), which is precisely colocalized with AChRs at synaptic sites from the earliest stages of neuromuscular synaptogenesis. In heterologous systems, expression of recombinant rapsyn leads to clustering of diffusely distributed AChRs, suggesting that rapsyn may control formation of clusters. To assess the role of rapsyn in vivo, we generated and characterized mutant mice with a targeted disruption of the Rapsyn gene. We report that rapsyn is essential for the formation of AChR clusters, but that synapse-specific transcription of AChR subunit genes can proceed in its absence.

Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels?

Polyclonal antibodies against each of the K+ channel subunits (Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2) shown previously to be expressed in adult rat heart at the mRNA level were used to examine the distributions of these K+ channel subunits in adult rat atrial and ventricular membranes. Immunohistochemistry on isolated adult rat ventricular myocytes revealed strong labeling with the anti-Kv4.2 and anti-Kv1.2 antibodies. Although somewhat weaker (than with anti-Kv1.2 or anti-Kv4.2), positive staining was also observed with the anti-Kv1.5 and anti-Kv2.1 antibodies. Ventricular myocytes exposed to the anti-Kv1.4 antibody, in contrast, did not appear significantly different from background. Qualitatively similar results were obtained on isolated adult rat atrial myocytes. Western blots of atrial and ventricular membrane proteins confirmed the presence of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 and revealed differences in the relative abundances of these subunits in the two membrane preparations. Kv4.2, for example, is more abundant in ventricular than in atrial membranes, whereas Kv1.2 and Kv2.1 are higher in atrial membranes; Kv1.5 levels are comparable in the two preparations. In contrast to these results, nothing was detected in Western blots of atrial or ventricular membrane proteins with the anti-Kv1.4 antibody at concentrations that revealed intense labeling of a 97-kD protein in adult rat brain membranes. A very faint band was detected at 97 kD in the atrial and ventricular preparations when the anti-Kv1.4 antibody was used at a 5- to 10-fold higher concentration. The simplest interpretation of these results is that Kv1.4 is not an abundant protein in adult rat atrial or ventricular myocytes. Therefore, it seems unlikely that Kv1.4 plays an important role in the formation of functional depolarization-activated K+ channels in these cells. The relation(s) between the (other four) K+ channel subunits and the depolarization-activated K+ channels identified electrophysiologically in adult rat atrial and ventricular myocytes is discussed in the present study.