Aldose reductase regulates TNF-alpha-induced cell signaling and apoptosis in vascular endothelial cells

TNF-alpha, generated during the systemic inflammatory response, triggers a wide range of biological activities that mediate the neurologic manifestations associated with cancer and infection. Since this cytokine regulates ion channels in vitro (especially Kv1.3 and Kir2.1), we aimed to study Kv1.3 and Kir2.1 expression in brain in response to in vivo systemic inflammation. Cancer-induced cachexia and LPS administration increased plasma TNF-alpha. Kv1.3 and Kir2.1 expression was impaired in brain during cancer cachexia. However, LPS treatment induced Kv1.3 and downregulated Kir2.1 expression, and TNF-alpha administration mimicked these results. Experiments using TNF-alpha double receptor knockout mice demonstrated that the systemic inflammatory response mediates K(+) channel regulation in brain via TNF-alpha-dependent and -independent redundant pathways. In summary, distinct neurological alterations associated with systemic inflammation may result from the interaction of various cytokine pathways tuning ion channel expression in response to neurophysiological and neuroimmunological processes.     

Behavioral motor dysfunction in Kv3-type potassium channel-deficient mice

The voltage-gated potassium channels Kv3.1 and Kv3.3 are expressed in several distinct neuronal subpopulations in brain areas known to be involved in motor control such as cortex, basal ganglia and cerebellum. Depending on the lack of Kv3.1 or Kv3.3 channel subunits, mutant mice show different Kv3-null allele-dependent behavioral alterations that include constitutive hyperactivity, sleep loss, impaired motor performance and, in the case of the Kv3.1/Kv3.3 double mutant, also severe ataxia, tremor and myoclonus (Espinosa et al. 2001, J Neurosci 21, 6657-6665, Genes, Brain Behav 3, 90-100). The lack of Kv3.1 channel subunits is mainly responsible for the constitutively increased locomotor activity and for sleep loss, whereas the absence of Kv3.3 subunits affects cerebellar function, in particular Purkinje cell discharges and olivocerebellar system properties (McMahon et al. 2004, Eur J Neurosci 19, 3317-3327). Here, we describe two sensitive and non-invasive tests to reliably quantify normal and abnormal motor functions, and we apply these tests to characterize motor dysfunction in Kv3-mutant mice. In contrast to wildtype and Kv3.1-single mutants, Kv3.3-single mutants and Kv3 mutants lacking three and four Kv3 alleles display Kv3-null allele-dependent gait alterations. Although the Kv3-null allele-dependent gait changes correlate with reduced motor performance, they appear to not affect the training-induced improvement of motor performance. These findings suggest that altered cerebellar physiology in the absence of Kv3.3 channels is responsible for impaired motor task execution but not motor task learning.     

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Mammalian brains contain relatively high amounts of common and uncommon sialylated N-glycan structures. Sialic acid linkages were identified for voltage-gated potassium channels, Kv3.1, 3.3, 3.4, 1.1, 1.2 and 1.4, by evaluating their electrophoretic migration patterns in adult rat brain membranes digested with various glycosidases. Additionally, their electrophoretic migration patterns were compared with those of NCAM (neural cell adhesion molecule), transferrin and the Kv3.1 protein heterologously expressed in B35 neuroblastoma cells. Metabolic labelling of the carbohydrates combined with glycosidase digestion reactions were utilized to show that the N-glycan of recombinant Kv3.1 protein was capped with an oligo/poly-sialyl unit. All three brain Kv3 glycoproteins, like NCAM, were terminated with alpha2,3-linked sialyl residues, as well as atypical alpha2,8-linked sialyl residues. Additionally, at least one of their antennae was terminated with an oligo/poly-sialyl unit, similar to recombinant Kv3.1 and NCAM. In contrast, brain Kv1 glycoproteins consisted of sialyl residues with alpha2,8-linkage, as well as sialyl residues linked to internal carbohydrate residues of the carbohydrate chains of the N-glycans. This type of linkage was also supported for Kv3 glycoproteins. To date, such a sialyl linkage has only been identified in gangliosides, not N-linked glycoproteins. We conclude that all six Kv channels (voltage-gated K+ channels) contribute to the alpha2,8-linked sialylated N-glycan pool in mammalian brain and furthermore that their N-glycan structures contain branched sialyl residues. Identification of these novel and unique sialylated N-glycan structures implicate a connection between potassium channel activity and atypical sialylated N-glycans in modulating and fine-tuning the excitable properties of neurons in the nervous system.     

Increased motor drive and sleep loss in mice lacking Kv3-type potassium channels

The voltage-gated potassium channels Kv3.1 and Kv3.3 are widely expressed in the brain, including areas implicated in the control of motor activity and in areas thought to regulate arousal states. Although Kv3.1 and Kv3.3-single mutants show some physiological changes, previous studies revealed relatively subtle behavioral alterations suggesting that Kv3.1 and Kv3.3 channel subunits may be encoded by a pair of redundant genes. In agreement with this hypothesis, Kv3.1/Kv3.3-deficient mice display a 'strong' mutant phenotype that includes motor dysfunction (ataxia, myoclonus, tremor) and hyperactivity when exposed to a novel environment. In this paper we report that Kv3.1/Kv3.3-deficient mice are also constitutively hyperactive. Compared to wildtype mice, double mutants display 'restlessness' that is particularly prominent during the light period, when mice are normally at rest, characterized by more than a doubling of ambulatory and stereotypic activity, and accompanied by a 40% sleep reduction. When we reinvestigated both single mutants, we observed constitutive increases of ambulatory and stereotypic activity in conjunction with sleep loss in Kv3.1-single mutants but not in Kv3.3-single mutants. These findings indicate that the absence of Kv3.1-channel subunits is primarily responsible for the increased motor drive and the reduction in sleep time.     

Heteromeric Kv1 potassium channel expression: amino acid determinants involved in processing and trafficking to the cell surface

Kv1.4 and Kv1.1 potassium channels are expressed in brain as mature glycoproteins that are trans-Golgi glycosylated. When expressed in cell lines these homomers had very different trans-Golgi glycosylation efficiencies and cell surface expression levels with Kv1.4 > Kv1.1 for both parameters (Zhu, J., Watanabe, I., Gomez, B., and Thornhill, W. B. (2001) J. Biol. Chem. 276, 39419-39427). This previous study identified determinants in the outer pore region of Kv1.4 and Kv1.1 that positively and negatively, respectively, affected these events when expressed as homomers. Here we investigated which subunit exhibited positive or negative effects on these processes when expressed as heteromers. Kv1.4/Kv1.1 heteromers, by coexpression or expression as tandem-linked heteromers, were expressed on the cell surface at approximately 20-fold lower levels versus Kv1.4 homomers but they were trans-Golgi glycosylated. The lower Kv1.4/Kv1.1 expression level was not rescued by Kvbeta 2.1 subunits. Thus Kv1.1 inhibited high cell surface expression and partially retained the heteromer in the endoplasmic reticulum, whereas Kv1.4 stimulated trans-Golgi glycosylation. The subunit determinants and cellular events responsible for these differences were investigated. In a Kv1.4/Kv1.1 heteromer, the Kv1.1 pore was a major negative determinant, and it inhibited high cell surface expression because it induced high partial endoplasmic reticulum retention and it decreased protein stability. Other Kv1.1 regions also inhibited high surface expression of heteromers. The Kv1.1 C terminus induced partial Golgi retention and contributed to a decreased protein stability, whereas the Kv1.1 N terminus contributed to only a decreased protein stability. Thus a neuron may regulate its cell surface K+ channel protein levels by different Kv1 subfamily homomeric and heteromeric combinations that affect intracellular retention characteristics and protein stability.     

Endogenous KCNE subunits govern Kv2.1 K+ channel activation kinetics in Xenopus oocyte studies

Kv2.1 is a voltage-gated potassium (Kv) channel that generates delayed rectifier currents in mammalian heart and brain. The biophysical properties of Kv2.1 and other ion channels have been characterized by functional expression in heterologous systems, and most commonly in Xenopus laevis oocytes. A number of previous oocyte-based studies of mammalian potassium channels have revealed expression-level-dependent changes in channel properties, leading to the suggestion that endogenous oocyte factors regulate channel gating. Here, we show that endogenous oocyte potassium channel KCNE ancillary subunits xMinK and xMiRP2 slow the activation of oocyte-expressed mammalian Kv2.1 channels two-to-fourfold. This produces a sigmoidal relationship between Kv2.1 current density and activation rate in oocyte-based two-electrode voltage clamp studies. The effect of endogenous xMiRP2 and xMinK on Kv2.1 activation is diluted at high Kv2.1 expression levels, or by RNAi knockdown of either endogenous subunit. RNAi knockdown of both xMiRP2 and xMinK eliminates the correlation between Kv2.1 expression level and activation kinetics. The data demonstrate a molecular basis for expression-level-dependent changes in Kv channel gating observed in heterologous expression studies.     

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.     

Differential recruitment of Kv1.4 and Kv4.2 to lipid rafts by PSD-95

The activity of voltage-gated potassium (Kv) channels, and consequently their influence on cellular functions, can be substantially altered by phosphorylation. Several protein kinases that modulate Kv channel activity are found in membrane subdomains known as lipid rafts, which are thought to organize signaling complexes in the cell. Thus, we asked whether Kv1.4 and Kv4.2, two channels with critical roles in excitable cells, are found in lipid rafts. Acylation can target proteins to raft regions; however, Kv channels are not acylated, and therefore, a different mechanism must exist to bring them into these membrane subdomains. Because both Kv1.4 and Kv4.2 interact with postsynaptic density protein 95 (PSD-95), which is acylated (specifically, palmitoylated), we examined whether PSD-95 can recruit these channels to lipid rafts. We found that a portion of Kv1.4 and Kv4.2 protein in rat brain membranes is raft-associated. Lipid raft patching and immunostaining confirmed that some Kv4.2 is in Thy-1-containing rafts in rat hippocampal neurons. Using a heterologous expression system, we determined that palmitoylation of PSD-95 was crucial to its localization to lipid rafts. We then assessed the contribution of PSD-95 to the raft association of these channels. Co-expression of PSD-95 increased the amount of Kv1.4, but not Kv4.2, in lipid rafts. Deleting the PSD-95 binding motif of Kv1.4 eliminated this recruitment, as did substituting a palmitoylation-deficient PSD-95 mutant. This work represents the first evidence that PSD-95 binding can recruit Kv channels into lipid rafts, a process that could facilitate interactions with the protein kinases that affect channel activity.     

Shared requirement for dynein function and intact microtubule cytoskeleton for normal surface expression of cardiac potassium channels

Potassium channels at the cardiomyocyte surface must eventually be internalized and degraded, and changes in cardiac potassium channel expression are known to occur during myocardial disease. It is not known which trafficking pathways are involved in the control of cardiac potassium channel surface expression, and it is not clear whether all cardiac potassium channels follow a common pathway or many pathways. In the present study we have surveyed the role of retrograde microtubule-dependent transport in modulating the surface expression of several cardiac potassium channels in ventricular myocytes and heterologous cells. The disruption of microtubule transport in rat ventricular myocytes with nocodazole resulted in significant changes in potassium currents. A-type currents were enhanced 1.6-fold at +90 mV, rising from control densities of 20.9 +/- 2.8 to 34.0 +/- 5.4 pA/pF in the nocodazole-treated cells, whereas inward rectifier currents were reduced by one-third, perhaps due to a higher nocodazole sensitivity of Kir channel forward trafficking. These changes in potassium currents were associated with a significant decrease in action potential duration. When expressed in heterologous human embryonic kidney (HEK-293) cells, surface expression of Kv4.2, known to substantially underlie A-type currents in rat myocytes, was increased by nocodazole, by the dynein inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, and by p50 overexpression, which specifically interferes with dynein motor function. Peak current density was 360 +/- 61.0 pA/pF in control cells and 658 +/- 94.5 pA/pF in cells overexpressing p50. The expression levels of Kv2.1, Kv3.1, human ether-a-go-go-related gene, and Kir2.1 were similarly increased by p50 overexpression in this system. Thus the regulation of potassium channel expression involves a common dynein-dependent process operating similarly on the various channels.     

Deletion of the Kv2.1 delayed rectifier potassium channel leads to neuronal and behavioral hyperexcitability

The Kv2.1 delayed rectifier potassium channel exhibits high‐level expression in both principal and inhibitory neurons throughout the central nervous system, including prominent expression in hippocampal neurons. Studies of in vitro preparations suggest that Kv2.1 is a key yet conditional regulator of intrinsic neuronal excitability, mediated by changes in Kv2.1 expression, localization and function via activity‐dependent regulation of Kv2.1 phosphorylation. Here we identify neurological and behavioral deficits in mutant (Kv2.1^?/?) mice lacking this channel. Kv2.1^?/? mice have grossly normal characteristics. No impairment in vision or motor coordination was apparent, although Kv2.1^?/? mice exhibit reduced body weight. The anatomic structure and expression of related Kv channels in the brains of Kv2.1^?/? mice appear unchanged. Delayed rectifier potassium current is diminished in hippocampal neurons cultured from Kv2.1^?/? animals. Field recordings from hippocampal slices of Kv2.1^?/? mice reveal hyperexcitability in response to the convulsant bicuculline, and epileptiform activity in response to stimulation. In Kv2.1^?/? mice, long‐term potentiation at the Schaffer collateral – CA1 synapse is decreased. Kv2.1^?/? mice are strikingly hyperactive, and exhibit defects in spatial learning, failing to improve performance in a Morris Water Maze task. Kv2.1^?/? mice are hypersensitive to the effects of the convulsants flurothyl and pilocarpine, consistent with a role for Kv2.1 as a conditional suppressor of neuronal activity. Although not prone to spontaneous seizures, Kv2.1^?/? mice exhibit accelerated seizure progression. Together, these findings suggest homeostatic suppression of elevated neuronal activity by Kv2.1 plays a central role in regulating neuronal network function .     

Alternative splicing regulates kv3.1 polarized targeting to adjust maximal spiking frequency

Synaptic inputs received at dendrites are converted into digital outputs encoded by action potentials generated at the axon initial segment in most neurons. Here, we report that alternative splicing regulates polarized targeting of Kv3.1 voltage-gated potassium (Kv) channels to adjust the input-output relationship. The spiking frequency of cultured hippocampal neurons correlated with the level of endogenous Kv3 channels. Expression of axonal Kv3.1b, the longer form of Kv3.1 splice variants, effectively converted slow-spiking young neurons to fast-spiking ones; this was not the case for Kv1.2 or Kv4.2 channel constructs. Despite having identical biophysical properties as Kv3.1b, dendritic Kv3.1a was significantly less effective at increasing the maximal firing frequency. This suggests a possible role of channel targeting in regulating spiking frequency. Mutagenesis studies suggest the electrostatic repulsion between the Kv3.1b N/C termini, created by its C-terminal splice domain, unmasks the Kv3.1b axonal targeting motif. Kv3.1b axonal targeting increased the maximal spiking frequency in response to prolonged depolarization. This finding was further supported by the results of local application of channel blockers and computer simulations. Taken together, our studies have demonstrated that alternative splicing controls neuronal firing rates by regulating the polarized targeting of Kv3.1 channels.     

MinK, MiRP1, and MiRP2 diversify Kv3.1 and Kv3.2 potassium channel gating

High frequency firing in mammalian neurons requires ultra-rapid delayed rectifier potassium currents generated by homomeric or heteromeric assemblies of Kv3.1 and Kv3.2 potassium channel alpha subunits. Kv3.1 alpha subunits can also form slower activating channels by coassembling with MinK-related peptide 2 (MiRP2), a single transmembrane domain potassium channel ancillary subunit. Here, using channel subunits cloned from rat and expressed in Chinese hamster ovary cells, we show that modulation by MinK, MiRP1, and MiRP2 is a general mechanism for slowing of Kv3.1 and Kv3.2 channel activation and deactivation and acceleration of inactivation, creating a functionally diverse range of channel complexes. MiRP1 also negatively shifts the voltage dependence of Kv3.1 and Kv3.2 channel activation. Furthermore, MinK, MiRP1, and MiRP2 each form channels with Kv3.1-Kv3.2 heteromers that are kinetically distinct from one another and from MiRP/homomeric Kv3 channels. The findings illustrate a mechanism for dynamic expansion of the functional repertoire of Kv3.1 and Kv3.2 potassium currents and suggest roles for these alpha subunits outside the scope of sustained rapid neuronal firing.     

Hypoxic preconditioning enhances bone marrow mesenchymal stem cell migration via Kv2.1 channel and FAK activation

Transplantation using stem cells including bone marrow mesenchymal stem cells (BMSCs) is emerging as a potential regenerative therapy after ischemic attacks in the heart and brain. The migration capability of transplanted cells is a critical cellular function for tissue repair. Based on our recent observations that hypoxic preconditioning (HP) has multiple benefits in improving stem cell therapy and that the potassium Kv2.1 channel acts as a promoter for focal adhesion kinase (FAK) activation and cell motility, the present investigation tested the hypothesis that HP treatment can increase BMSC migration via the mechanism of increased Kv2.1 expression and FAK activities. BMSCs derived from green fluorescent protein-transgenic mice were treated under either normoxic (N-BMSC) or hypoxic (0.5% O(2)) (HP-BMSC) conditions for 24 h. Western blot analysis showed HP selectively upregulated Kv2.1 expression while leaving other K(+) channels, such as Kv1.5 and Kv1.4, unaffected. Compared with normoxic controls, significantly larger outward delayed rectifier K(+) currents were recorded in HP-BMSCs. HP enhanced BMSC migration/homing activities in vitro and after intravenous transplantation into rats subjected to permanent myocardial infarction (MI). The HP-promoted BMSC migration was inhibited by either blocking K(+) channels or knocking down Kv2.1. Supporting a relationship among HP, Kv2.1, and FAK activation, HP increased phosphorylation of FAK(397) and FAK(576/577), and this effect was antagonized by blocking K(+) channels. These findings provide novel evidence that HP enhances the ability of BMSCs to migrate and home to the injured region; this effect is mediated through a regulatory role of Kv2.1 on FAK phosphorylation/activation.     

Biochemical characterization of the native Kv2.1 potassium channel

Functional diversity of potassium channels in both prokaryotic and eukaryotic cells suggests multiple levels of regulation. Posttranslational regulation includes differential subunit assembly of homologous pore-forming subunits. In addition, a variety of modulatory subunits may interact with the pore complex either statically or dynamically. Kv2.1 is a delayed rectifier potassium channel isolated by expression cloning. The native polypeptide has not been purified, hence composition of the Kv2.1 channel complexes was not well understood. Here we report a biochemical characterization of Kv2.1 channel complexes from both recombinant cell lines and native rat brain. The channel complexes behave as large macromolecular complexes with an apparent oligomeric size of 650 kDa as judged by gel filtration chromatography. The molecular complexes have distinct biochemical populations detectable by a panel of antibodies. This is indicative of functional heterogeneity. Despite mRNA distribution in a variety of tissues, the native Kv2.1 polypeptides are more abundantly found in brain and have predominantly Kv2.1 subunits but not homologous Kv2.2 subunits. The proteins precipitated by anti-Kv2.1 and their physiological relevance are of interest for further investigation.     

Homology model and targeted mutagenesis identify critical residues for arachidonic acid inhibition of Kv4 channels

Polyunsaturated fatty acids such as arachidonic acid (AA) exhibit inhibitory modulation of Kv4 potassium channels. Molecular docking approaches using a Kv4.2 homology model predicted a membrane-embedded binding pocket for AA comprised of the S4-S5 linker on one subunit and several hydrophobic residues within S3, S5 and S6 from an adjacent subunit. The pocket is conserved among Kv4 channels. We tested the hypothesis that modulatory effects of AA on Kv4.2/KChIP channels require access to this site. Targeted mutation of a polar residue (K318) and a nonpolar residue (G314) within the S4-S5 linker as well as a nonpolar residue in S3 (V261) significantly impaired the effects of AA on K⁺ currents in Xenopus oocytes. These residues may be important in stabilizing (K318) or regulating access to (V261, G314) the negatively charged carboxylate moiety on the fatty acid. Structural specificity was supported by the lack of disruption of AA effects observed with mutations at residues located near, but not within the predicted binding pocket. Furthermore, we found that the crystal structure of the related Kv1.2/2.1 chimera lacks the structural features present in the proposed AA docking site of Kv4.2 and the Kv1.2/2.1 K⁺ currents were unaffected by AA. We simulated the mutagenic substitutions in our Kv4.2 model to demonstrate how specific mutations may disrupt the putative AA binding pocket. We conclude that AA inhibits Kv4 channel currents and facilitates current decay by binding within a hydrophobic pocket in the channel in which K318 within the S4-S5 linker is a critical residue for AA interaction.     

KCNE1 and KCNE2 inhibit forward trafficking of homomeric N-type voltage-gated potassium channels

Potassium currents generated by voltage-gated potassium (Kv) channels comprising α-subunits from the Kv1, 2, and 3 subfamilies facilitate high-frequency firing of mammalian neurons. Within these subfamilies, only three α-subunits (Kv1.4, Kv3.3, and Kv3.4) generate currents that decay rapidly in the open state because an N-terminal ball domain blocks the channel pore after activation—a process termed N-type inactivation. Despite its importance to shaping cellular excitability, little is known of the processes regulating surface expression of N-type α-subunits, versus their slowly inactivating (delayed rectifier) counterparts. Here we found that currents generated by homomeric Kv1.4, Kv3.3, and Kv3.4 channels are all strongly suppressed by the single transmembrane domain ancillary (β) subunits KCNE1 and KCNE2. A combination of electrophysiological, biochemical, and immunofluorescence analyses revealed this suppression is due to KCNE1 and KCNE2 retaining Kv1.4 and Kv3.4 intracellularly, early in the secretory pathway. The retention is specific, requires α-β coassembly, and does not involve the dynamin-dependent endocytosis pathway. However, the small fraction of Kv3.4 that escapes KCNE-dependent retention is regulated by dynamin-dependent endocytosis. The findings illustrate two contrasting mechanisms controlling surface expression of N-type Kv α-subunits and therefore, potentially, cellular excitability and refractory periods.     

Precise localization of the voltage-gated potassium channel subunits Kv3.1b and Kv3.3 revealed in the molecular layer of the rat cerebellar cortex by a pre-embedding immunogold method

A proper motor activity relies on a correct cerebellar function. The Kv3.1 and Kv3.3 voltage-gated potassium channels are key proteins involved in cerebellar function and dysfunction, as the lack of these causes severe motor deficits. Both channel subunits are coexpressed in granule cells and are rapidly activated at relatively positive potentials to support the generation of fast action potentials. However, the contribution of each subunit to the molecular architecture of the parallel fibers, the granule cell axons, is so far unknown. The goal of this study was to elucidate the relative distribution of Kv3.1b and Kv3.3 in specific compartments of the rat parallel fibers by using a pre-embedding immunocytochemical method for electron microscopy. Numerous Kv3.1b and Kv3.3 silver-intensified gold particles were associated with membranes of parallel fiber synaptic terminals and their intervaricose segments. Kv3.1b was found in about 85% of parallel fiber synaptic terminals and in about 47% of their intervaricose portions. However, only 28% of intervaricosities and 23% of parallel fiber presynaptic boutons were Kv3.3 immunopositive. The analysis also revealed that 54% of Purkinje cell dendritic spines localized Kv3.3. Although both potassium channel subunits share localization in the same presynaptic parallel fiber compartments, the present results with the method used indicate that there are a higher percentage of parallel fibers labeled for Kv3.1b than for Kv3.3, and that the labeling intensity for each subunit is higher in specific subcompartments analyzed than in others.