Shaker-type Potassium Channels Research Articles

LGI3/2-ADAM23 interactions cluster Kv1 channels in myelinated axons to regulate refractory period.

Along myelinated axons, Shaker-type potassium channels (Kv1) accumulate at high density in the juxtaparanodal region, directly adjacent to the paranodal axon-glia junctions that flank the nodes of Ranvier. However, the mechanisms that control the clustering of Kv1 channels, as well as their function at this site, are still poorly understood. Here we demonstrate that axonal ADAM23 is essential for both the accumulation and stability of juxtaparanodal Kv1 complexes. The function of ADAM23 is critically dependent on its interaction with its extracellular ligands LGI2 and LGI3. Furthermore, we demonstrate that juxtaparanodal Kv1 complexes affect the refractory period, thus enabling high-frequency burst firing of action potentials. Our findings not only reveal a previously unknown molecular pathway that regulates Kv1 channel clustering, but they also demonstrate that the juxtaparanodal Kv1 channels that are concealed below the myelin sheath, play a significant role in modifying axonal physiology.

Functional role of endogenous Kv1.4 in experimental demyelination

During neuroinflammation, the shaker type potassium channel Kv1.4 is re-expressed in oligodendrocytes (Ol), but not immune cells. Here, we analyze the role of endogenous Kv1.4 in two demyelinating animal models of multiple sclerosis. While Kv1.4 deficiency in primary murine Ol led to a decreased proliferation rate in vitro, it did not exert an effect on Ol proliferation or on the extent of de- or remyelination in the cuprizone model in vivo. However, in experimental autoimmune encephalomyelitis, Kv1.4−/− mice exhibited a milder disease course and reduced Th1 responses. These data argue for an indirect effect of Kv1.4 on immune cells, possibly via glial cells.

C. elegans AWA Olfactory Neurons Fire Calcium-Mediated All-or-None Action Potentials

Neurons in Caenorhabditis elegans and other nematodes have been thought to lack classical action potentials. Unexpectedly, we observe membrane potential spikes with defining characteristics of action potentials in C.elegans AWA olfactory neurons recorded under current-clamp conditions. Ion substitution experiments, mutant analysis, pharmacology, and modeling indicate that AWA fires calcium spikes, which are initiated by EGL-19 voltage-gated CaV1 calcium channels and terminated by SHK-1 Shaker-type potassium channels. AWA action potentials result in characteristic signals in calcium imaging experiments. These calcium signals are also observed when intact animals are exposed to odors, suggesting that natural odor stimuli induce AWA spiking. The stimuli that elicit action potentials match AWA's specialized function in climbing odor gradients. Our results provide evidence that C.elegans neurons can encode information through regenerative all-or-none action potentials, expand the computational repertoire of its nervous system, and inform future modeling of its neural coding and network dynamics.

Ion Channels Regulate Nyctinastic Leaf Opening in Samanea saman

The circadian leaf opening and closing (nyctinasty) of Fabaceae has attracted scientists' attention since the era of Charles Darwin. Nyctinastic movement is triggered by the alternate swelling and shrinking of motor cells at the base of the leaf. This, in turn, is facilitated by changing osmotic pressures brought about by ion flow through anion and potassium ion channels. However, key regulatory ion channels and molecular mechanisms remain largely unknown. Here, we identify three key ion channels in mimosoid tree Samanea saman: the slow-type anion channels, SsSLAH1 and SsSLAH3, and the Shaker-type potassium channel, SPORK2. We show that cell-specific circadian expression of SsSLAH1 plays a key role in nyctinastic leaf opening. In addition, SsSLAH1 co-expressed with SsSLAH3 in flexor (abaxial) motor cells promoted leaf opening. We confirm the importance of SLAH1 in leaf movement using SLAH1-impaired Glycine max. Identification of this "master player" advances our molecular understanding of nyctinasty.

HRPU-2, a Homolog of Mammalian hnRNP U, Regulates Synaptic Transmission by Controlling the Expression of SLO-2 Potassium Channel in Caenorhabditis elegans.

Slo2 channels are large-conductance potassium channels abundantly expressed in the nervous system. However, it is unclear how their expression level in neurons is regulated. Here we report that HRPU-2, an RNA-binding protein homologous to mammalian heterogeneous nuclear ribonucleoprotein U (hnRNP U), plays an important role in regulating the expression of SLO-2 (a homolog of mammalian Slo2) in Caenorhabditis elegans Loss-of-function (lf) mutants of hrpu-2 were isolated in a genetic screen for suppressors of a sluggish phenotype caused by a hyperactive SLO-2. In hrpu-2(lf) mutants, SLO-2-mediated delayed outward currents in neurons are greatly decreased, and neuromuscular synaptic transmission is enhanced. These mutant phenotypes can be rescued by expressing wild-type HRPU-2 in neurons. HRPU-2 binds to slo-2 mRNA, and hrpu-2(lf) mutants show decreased SLO-2 protein expression. In contrast, hrpu-2(lf) does not alter the expression of either the BK channel SLO-1 or the Shaker type potassium channel SHK-1. hrpu-2(lf) mutants are indistinguishable from wild type in gross motor neuron morphology and locomotion behavior. Together, these observations suggest that HRPU-2 plays important roles in SLO-2 function by regulating SLO-2 protein expression, and that SLO-2 is likely among a restricted set of proteins regulated by HRPU-2. Mutations of human Slo2 channel and hnRNP U are strongly linked to epileptic disorders and intellectual disability. The findings of this study suggest a potential link between these two molecules in human patients.SIGNIFICANCE STATEMENT Heterogeneous nuclear ribonucleoprotein U (hnRNP U) belongs to a family of RNA-binding proteins that play important roles in controlling gene expression. Recent studies have established a strong link between mutations of hnRNP U and human epilepsies and intellectual disability. However, it is unclear how mutations of hnRNP U may cause such disorders. This study shows that mutations of HRPU-2, a worm homolog of mammalian hnRNP U, result in dysfunction of a Slo2 potassium channel, which is critical to neuronal function. Because mutations of Slo2 channels are also strongly associated with epileptic encephalopathies and intellectual disability in humans, the findings of this study point to a potential mechanism underlying neurological disorders caused by hnRNP U mutations.

Co-cultures with stem cell-derived human sensory neurons reveal regulators of peripheral myelination.

See Saporta and Shy (doi:10.1093/awx048) for a scientific commentary on this article.Effective bidirectional signalling between axons and Schwann cells is essential for both the development and maintenance of peripheral nerve function. We have established conditions by which human induced pluripotent stem cell-derived sensory neurons can be cultured with rat Schwann cells, and have produced for the first time long-term and stable myelinating co-cultures with human neurons. These cultures contain the specialized domains formed by axonal interaction with myelinating Schwann cells, such as clustered voltage-gated sodium channels at the node of Ranvier and Shaker-type potassium channel (Kv1.2) at the juxtaparanode. Expression of type III neuregulin-1 (TIIINRG1) in induced pluripotent stem cell-derived sensory neurons strongly enhances myelination, while conversely pharmacological blockade of the NRG1-ErbB pathway prevents myelination, providing direct evidence for the ability of this pathway to promote the myelination of human sensory axons. The β-secretase, BACE1 is a protease needed to generate active NRG1 from the full-length form. Due to the fact that it also cleaves amyloid precursor protein, BACE1 is a therapeutic target in Alzheimer's disease, however, consistent with its role in NRG1 processing we find that BACE1 inhibition significantly impairs myelination in our co-culture system. In order to exploit co-cultures to address other clinically relevant problems, they were exposed to anti-disialosyl ganglioside antibodies, including those derived from a patient with a sensory predominant, inflammatory neuropathy with mixed axonal and demyelinating electrophysiology. The co-cultures reveal that both mouse and human disialosyl antibodies target the nodal axolemma, induce acute axonal degeneration in the presence of complement, and impair myelination. The human, neuropathy-associated IgM antibody is also shown to induce complement-independent demyelination. Myelinating co-cultures using human induced pluripotent stem cell-derived sensory neurons thus provide insights into the cellular and molecular specialization of axoglial signalling, how pharmacological agents may promote or impede such signalling and the pathogenic effects of ganglioside antibodies.awx012media15372351982001.

Structural Analysis and Deletion Mutagenesis Define Regions of QUIVER/SLEEPLESS that Are Responsible for Interactions with Shaker-Type Potassium Channels and Nicotinic Acetylcholine Receptors.

Ly6 proteins are endogenous prototoxins found in most animals. They show striking structural and functional parallels to snake α-neurotoxins, including regulation of ion channels and cholinergic signaling. However, the structural contributions of Ly6 proteins to regulation of effector molecules is poorly understood. This question is particularly relevant to the Ly6 protein QUIVER/SLEEPLESS (QVR/SSS), which has previously been shown to suppress excitability and synaptic transmission by upregulating potassium (K) channels and downregulating nicotinic acetylcholine receptors (nAChRs) in wake-promoting neurons to facilitate sleep in Drosophila. Using deletion mutagenesis, co-immunoprecipitations, ion flux assays, surface labeling and confocal microscopy, we demonstrate that only loop 2 is required for many of the previously described properties of SSS in transfected cells, including interactions with K channels and nAChRs. Collectively our data suggest that QVR/SSS, and by extension perhaps other Ly6 proteins, target effector molecules using limited protein motifs. Mapping these motifs may be useful in rational design of drugs that mimic or suppress Ly6-effector interactions to modulate nervous system function.

Inhalational anesthetics disrupt postsynaptic density protein-95, Drosophila disc large tumor suppressor, and zonula occludens-1 domain protein interactions critical to action of several excitatory receptor channels related to anesthesia.

The authors have shown previously that inhaled anesthetics disrupt the interaction between the second postsynaptic density protein-95, Drosophila disc large tumor suppressor, and zonula occludens-1 (PDZ) domain of postsynaptic density protein-95 (PSD-95) and the C-terminus of N-methyl-D-aspartate receptor subunits NR2A and NR2B. The study data indicate that PDZ domains may serve as a molecular target for inhaled anesthetics. However, the underlying molecular mechanisms remain to be illustrated. Glutathione S-transferase pull-down assay, coimmunoprecipitation, and yeast two-hybrid analysis were used to assess PDZ domain-mediated protein-protein interactions in different conditions. Nuclear magnetic resonance spectroscopy was used to investigate isoflurane-induced chemical shift changes in the PDZ1-3 domains of PSD-95. A surface plasmon resonance-based BIAcore (Sweden) assay was used to examine the ability of isoflurane to inhibit the PDZ domain-mediated protein-protein interactions in real time. Halothane and isoflurane dose-dependently inhibited PDZ domain-mediated interactions between PSD-95 and Shaker-type potassium channel Kv1.4 and between α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluA2 and its interacting proteins-glutamate receptor-interacting protein or protein interacting with c kinase 1. However, halothane and isoflurane had no effect on PDZ domain-mediated interactions between γ-aminobutyric acid type B receptor and its interacting proteins. The inhaled anesthetic isoflurane mostly affected the residues close to or in the peptide-binding groove of PSD-95 PDZ1 and PDZ2 (especially PDZ2), while barely affecting the peptide-binding groove of PSD-95 PDZ3. These results suggest that inhaled anesthetics interfere with PDZ domain-mediated protein-protein interactions at several receptors important to neuronal excitation, anesthesia, and pain processing.

Potassium channel regulator KCNRG regulates surface expression of Shaker-type potassium channels

Besides their role in the generation of action potentials, voltage-gated potassium channels are implicated in cellular processes ranging from cell division to cell death. The K + channel regulator protein (KCNRG), identified as a putative tumor suppressor, reduces K + currents through human K + channels hKv1.1 and hKv1.4 expressed in Xenopus oocytes. Current attenuation requires the presence of the N-terminal T1 Domain and immunoprecipitation experiments suggest association of KCNRG with the N-terminus of the channel. Our data indicates that KCNRG is an ER-associated protein, which we propose regulates Kv1 family channel proteins by retaining a fraction of channels in endomembranes.

Plant ion channels: gene families, physiology, and functional genomics analyses.

Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization- and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide-gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport.

Remyelination of dorsal column axons by endogenous Schwann cells restores the normal pattern of Nav1.6 and Kv1.2 at nodes of Ranvier

Demyelination of CNS axons occurs in a number of pathological conditions, including multiple sclerosis and contusion-type spinal cord injury. The demyelination can be repaired by remyelination in both humans and rodents, and even within the CNS remyelination can be achieved by endogenous and/or exogenous Schwann cells, the myelinating cells of the PNS. Remyelinated axons can often conduct impulses securely, but the organization of ion channels at long-term remyelinated nodes is not known. In the present study, the expression of voltage-gated sodium (Na(v)) and potassium (K(v)) channels along central axons remyelinated by endogenous Schwann cells has been studied in lesions induced more than 1 year previously by the intraspinal injection of ethidium bromide (EB). The expression of the channels at long-term nodes formed by Schwann cell remyelination has been compared with that present in nascent nodes formed in the adult at 18 and 23 days post-EB injection. Immunohistochemical studies revealed that long-term nodes formed by Schwann cell remyelination exhibit a clustering of Na(v)1.6 sodium channels within the nodal membrane, with the Shaker-type potassium channel K(v)1.2 segregated within the juxtaparanodal region, similar to the arrangement at normal mature CNS nodes. Na(v)1.2 was not detected at nodes formed by Schwann cells at any stage of their development. Moreover, Na(v)1.6, but not Na(v)1.2, was clustered at nascent nodes formed by remyelinating Schwann cells 18 and 23 days following EB injection. These observations show that endogenous Schwann cells can establish and maintain nodes of Ranvier on central axons for over one year, and that the nodes exhibit an apparently normal distribution of sodium and potassium channels, with Na(v)1.6 the predominant subtype of sodium channel present at such nodes at all stages of their development.

Abbreviated action potential kinetics in a mouse model of potassium channel overexpression during hippocampal development.

Loss or "gain" of function mutations in voltage-gated ion channels often results in an adverse neurological phenotype. We have examined the electrical characteristics of hippocampal pyramidal cells in a transgenic mouse model to determine how overexpression of a Shaker-type potassium channel subunit during early postnatal development might alter excitability properties of developing neurons. We found that in CA3 neurons potassium channel overexpression led to a transient shortening in duration of single action potentials during the first two postnatal weeks. There was an increase in maximal repolarization rate, without significant effect on the rate of rise. Transgenic CA3 neurons also showed a decrease of firing frequency in response to sustained depolarizing current injection. In contrast, repolarization of action potentials in CA1 neurons was not significantly altered by trangene expression. Western Blot Analysis of membrane-associated transgene protein indicated that transgene protein levels decreased during development, in agreement with functional measures of spike width. Our data indicate that the functional consequences of potassium channel transgene expression are dependent on cellular environment and developmental stage. A transient period of hypoexcitability during a critical period of development for CA3 neurons may contribute to the hyperexcitable phenotype observed in adult animals.

ZIP3, a New Splice Variant of the PKC-ζ-interacting Protein Family, Binds to GABAC Receptors, PKC-ζ, and Kvβ2

The correct targeting of modifying enzymes to ion channels and neurotransmitter receptors represents an important biological mechanism to control neuronal excitability. The recent cloning of protein kinase C-zeta interacting proteins (ZIP1, ZIP2) identified new scaffolds linking the atypical protein kinase PKC-zeta to target proteins. GABA(C) receptors are composed of three rho subunits (rho 1-3) that are highly expressed in the retina, where they are clustered at synaptic terminals of bipolar cells. A yeast two-hybrid screen for the GABA(C) receptor rho 3 subunit identified ZIP3, a new C-terminal splice variant of the ZIP protein family. ZIP3 was ubiquitously expressed in non-neuronal and neuronal tissues, including the retina. The rho 3-binding region of ZIP3 contained a ZZ-zinc finger domain, which interacted with 10 amino acids conserved in rho 1-3 but not in GABA(A) receptors. Consistently, only rho 1-3 subunits bound to ZIP3. ZIP3 formed dimers with ZIP1-3 and interacted with PKC-zeta and the shaker-type potassium channel subunit Kv beta 2. Different domains of ZIP3 interacted with PKC-zeta and the rho 3 subunit, and simultaneous assembly of ZIP3, PKC-zeta and rho 3 was demonstrated in vitro. Subcellular co-expression of ZIP3 binding partners in the retina supported the proposed protein interactions. Our results indicate the formation of a ternary postsynaptic complex containing PKC-zeta, ZIP3, and GABA(C) receptors.

Protein 4.1B associates with both Caspr/paranodin and Caspr2 at paranodes and juxtaparanodes of myelinated fibres.

Caspr/paranodin, a neuronal transmembrane glycoprotein, is essential for the structure and function of septate-like paranodal axoglial junctions at nodes of Ranvier. A closely related protein, Caspr2, is concentrated in juxtaparanodal regions where it associates indirectly with the shaker-type potassium channels. Although ultrastructural studies indicate that paranodal complexes are linked to the cytoskeleton, the intracellular partners of Caspr/paranodin, as well as those of Caspr2, are poorly characterized. We show that the conserved intracellular juxtamembrane regions (GNP motif) of Caspr/paranodin and Caspr2 bind proteins 4.1R and 4.1B. 4.1B is known to be enriched in paranodal and juxtaparanodal regions. 4.1B immunoreactivity accumulates progressively at paranodes and juxtaparanodes during postnatal development, following the concentration of Caspr/paranodin and Caspr2, respectively, in central and peripheral myelinated axons. These two proteins coimmunoprecipitated with 4.1B in brain homogenates. Our results provide strong evidence for the association of 4.1B with Caspr/paranodin at paranodes and with Caspr2 at juxtaparanodes. We propose that 4.1B anchors these axonal proteins to the actin-based cytoskeleton in these two regions.

Regulation of Shaker-type potassium channels by hypoxia. Oxygen-sensitive K+ channels in PC12 cells.

Little is known about the molecular composition of the O2-sensitive K+ (Ko2) channels. The possibility that these channels belong to the Shaker subfamily (Kv1) of voltage-dependent K+ (Kv) channels has been raised in pulmonary artery (PA) smooth muscle cells. Numerous findings suggest that the Ko2 channel in PC12 cells is a Kv1 channel, formed by the Kv1.2 alpha subunit. The Ko2 channel in PC12 cells is a slow-inactivating voltage-dependent K+ channel of 20 pS conductance. Other Kv channels, also expressed in PC12 cells, are not inhibited by hypoxia. Selective up-regulation by chronic hypoxia of the Kv1.2 alpha subunit expression correlates with an increase O2-sensitivity of the K+ current. Other Kv1 alpha subunit genes encoding slow-inactivating Kv channels, such as Kv1.3, Kv2.1, Kv3.1 and Kv3.2 are not modulated by chronic hypoxia. The Ko2 current in PC12 cells is blocked by 5 mM externally applied tetraethylammonium chloride (TEA) and by charydbotoxin (CTX). The responses of the Kv1.2 K+ channel to hypoxia have been studied in the Xenopus oocytes and compared to those of Kv2.1, also proposed as Ko2 channel in PA smooth muscle cells. Two-electrode voltage clamp experiments show that hypoxia induces inhibition of K+ current amplitude only in oocytes injected with Kv1.2 cRNA. These data indicate that Kv1.2 K+ channels are inhibited by hypoxia.

Internodal specializations of myelinated axons in the central nervous system.

We have examined the localization of contactin-associated protein (Caspr), the Shaker-type potassium channels, Kv1.1 and Kv1.2, their associated beta subunit, Kvbeta2, and Caspr2 in the myelinated fibers of the CNS. Caspr is localized to the paranodal axonal membrane, and Kv1.1, Kv1.2, Kvbeta2 and Caspr2 to the juxtaparanodal membrane. In addition to the paranodal staining, an internodal strand of Caspr staining apposes the inner mesaxon of the myelin sheath. Unlike myelinated axons in the peripheral nervous system, there was no internodal strand of Kv1.1, Kv1.2, Kvbeta2, or Caspr2. Thus, the organization of the nodal, paranodal, and juxtaparanodal axonal membrane is similar in the central and peripheral nervous systems, but the lack of Kv1.1/Kv1.2/Kvbeta2/Caspr2 internodal strands indicates that the oligodendrocyte myelin sheaths lack a trans molecular interaction with axons, an interaction that is present in Schwann cell myelin sheaths.

The genomic basis of K V3.4 potassium channel mRNA diversity in mice

K V3.4 belongs to the shaw subfamily of shaker-type potassium channels. It conducts fast inactivating, high threshold currents in the central nervous system and in fast-twitch skeletal muscle fibers. The corresponding mouse gene, Kcnc4, consists of five exons spanning a region of 20 kb. Approximately 700 bp of regulatory sequence were delineated. It is GC-rich and lacks typical TATA and CAAT motifs. Instead, seven Sp-1 and three E-box elements define putative regulatory sequences. The mouse K V3.4 mRNA has a size of 3639 bp, 1120 bp of which are 3′ untranslated region. A transcript initiated from an alternative 5′-exon was identified by RACE and verified by genomic analysis. This isoform, designated K V3.4d, is predominantly expressed in skeletal muscle and probably results from alternative promoter usage. It encodes a channel protein with a novel N-terminal cytoplasmic domain. It lacks the conserved sequence motifs encoding the shaw-type tetramerization domain and the ‘ball’ peptide, which confers fast inactivation properties. Another splice variant, K V3.4c, is derived by exon skipping in the C-terminal region and is expressed at similar levels in brain and muscle. These data demonstrate that differential splicing and alternative transcription start sites are utilised to generate a set of K V3.4 variants in skeletal muscle and brain, presumably involved in the regulation of excitability.

Improved immunohistochemical detection of postsynaptically located PSD‐95/SAP90 protein family by protease section pretreatment: A study in the adult mouse brain

Postsynaptic density (PSD)–95, SAP102, and Chapsyn-110 are members of the PSD-95/SAP90 protein family, which interact with the C-terminus of N-methyl-D-aspartate (NMDA) receptor and shaker-type potassium channel subunits. Here we report that appropriate section pretreatment with pepsin has led to qualitative and quantitative changes in light microscopic immunohistochemical detection of the protein family. First, pepsin pretreatment lowered the concentration of affinity-purified primary antibodies, while it greatly increased the intensity of immunoreactions. Second, the resulting overall distributions of PSD-95, SAP102, and Chapsyn-110 in the adult mouse brain were consistent with their mRNA distributions. Third, instead of the reported patterns of somatodendritic labeling, tiny punctate staining in the neuropil became overwhelming. Fourth, many PSD-95-immunopositive puncta were apposed closely to synaptophysin-positive nerve terminals and overlapped with NMDA receptor subunits. By postembedding immunogold, the PSD-95 antibody was shown to label exclusively the postsynaptic density at asymmetrical synapses. Based on these results, we conclude that antibody access and binding to the postsynaptically located PSD-95/SAP90 protein family are hindered when conventional immunohistochemistry is adopted, and that pepsin pretreatment effectively unmasks the postsynaptic epitopes. On the other hand, PSD-95 in axon terminals of cerebellar basket cells, where high levels of potassium channels are present, was detectable irrespective of pepsin pretreatment, suggesting that PSD-95 antibody is readily accessible to the presynaptic epitopes. Consequently, the present immunohistochemical results have provided light microscopic evidence supporting the prevailing notion that the PSD-95/SAP90 protein family interacts with NMDA receptor subunits and potassium channel subunits. J. Comp. Neurol. 426:572–586, 2000. © 2000 Wiley-Liss, Inc.

Cytoplasmic residues influence the voltage-dependence of the gating of human K+ channels.

Shaker type potassium channels are strongly voltage dependent and potassium selective. Kv1.4 channels from a variety of sources exhibit a much shallower voltage-dependence of activation than other members of the family. We have made a chimeric construct consisting of the N-terminal chain of hKv1.1 spliced onto the transmembrane portion of hKv1.4 (IN/4). When expressed in Xenopus oocytes, the chimeric channel exhibits a voltage dependence that is similar to hKv1.1 although the voltage sensing and transduction machinery presumably reside in the transmembrane portion of the channel. Loss of the N-terminal ball and chain from hKv1.4 is not responsible for this as a truncation construct, starting close to the splice junction, has the same voltage-dependence as full length hKv1.4. We suggest that residues from the N-terminal chain of hKv1.1 interact with the machinery that transduces movement of the voltage sensor into channel opening. If so, this chimeric construct could provide a handle to the identification of elements of this transduction machinery.

Improved immunohistochemical detection of postsynaptically located PSD-95/SAP90 protein family by protease section pretreatment: A study in the adult mouse brain

Postsynaptic density (PSD)-95, SAP102, and Chapsyn-110 are members of the PSD-95/SAP90 protein family, which interact with the C-terminus of N-methyl-D-aspartate (NMDA) receptor and shaker-type potassium channel subunits. Here we report that appropriate section pretreatment with pepsin has led to qualitative and quantitative changes in light microscopic immunohistochemical detection of the protein family. First, pepsin pretreatment lowered the concentration of affinity-purified primary antibodies, while it greatly increased the intensity of immunoreactions. Second, the resulting overall distributions of PSD-95, SAP102, and Chapsyn-110 in the adult mouse brain were consistent with their mRNA distributions. Third, instead of the reported patterns of somatodendritic labeling, tiny punctate staining in the neuropil became overwhelming. Fourth, many PSD-95-immunopositive puncta were apposed closely to synaptophysin-positive nerve terminals and overlapped with NMDA receptor subunits. By postembedding immunogold, the PSD-95 antibody was shown to label exclusively the postsynaptic density at asymmetrical synapses. Based on these results, we conclude that antibody access and binding to the postsynaptically located PSD-95/SAP90 protein family are hindered when conventional immunohistochemistry is adopted, and that pepsin pretreatment effectively unmasks the postsynaptic epitopes. On the other hand, PSD-95 in axon terminals of cerebellar basket cells, where high levels of potassium channels are present, was detectable irrespective of pepsin pretreatment, suggesting that PSD-95 antibody is readily accessible to the presynaptic epitopes. Consequently, the present immunohistochemical results have provided light microscopic evidence supporting the prevailing notion that the PSD-95/SAP90 protein family interacts with NMDA receptor subunits and potassium channel subunits.