Nodal Na(+)-channel displacement is associated with nerve-conduction slowing in the chronically diabetic BB/W rat: prevention by aldose reductase inhibition.

Chronic nerve conduction showing in experimental diabetic neuropathy has been associated with decreased nodal Na+ permeability and an ultrastructurally identifiable loss of axo-glial junctions, which comprise the paranodal voltage channel barrier separating nodal Na+ channels from paranodal K+ channels. In human and experimental diabetic neuropathy these structural changes of the paranodal apparatus correlate closely with the nerve conduction defect. The present immunocytochemical study of the alpha-subunit of the Na+ channel examined whether the breach of the voltage channel barrier may account for a shift in the distribution of Na+ channels explaining decreased nodal Na+ permeability. Biobreeding Wistar (BB/W) rats diabetic for 4-8 months showed a progressive redistribution of nodal Na+ channels across the paranodal barrier into the paranodal and internodal domains which was associated with chronic nerve conduction slowing. The present data suggest that structural damage to the paranodal barrier system in diabetic nerve facilitates the lateral displacement of Na+ channels from the nodal axolemma thereby diminishing their nodal density and the nodal Na+ permeability associated with the chronic nerve conduction defect in experimental diabetes. These abnormalities were prevented by the treatment with an aldose reductase inhibitor, belonging to a class of drugs that, in neuropathic patients, improves nerve-conduction velocity and repairs axo-glial dysjunction of the paranodal apparatus.

Addition of tetrodotoxin alters the morphology of thalamocortical axons in organotypic cocultures.

Living organotypic cocultures of rat thalamic and cortical explants were used to examine the effects of blocking action potential activity on the morphological development of axons in the mammalian neocortex. Studies in vivo have suggested that blocking sodium channel-dependent activity influences the growth characteristics of thalamocortical axons during development. We have extended these observations by using an in vitro system that affords more direct observational analysis of the early events of axonal growth in an accessible cellular environment DiI-labeled thalamocortical axons grow exuberantly into the target cortex and establish axonal connections that reflect the events of early thalamocortical afferent development. Within these cocultures, the morphological features of DiI-labeled axons can be readily distinguished. Tracings of thalamocortical axons were quantitated with respect to number, length, and termination pattern of axonal branches, as well as number of varicosities. Addition of the voltage-dependent sodium channel blocker, tetrodotoxin, to cocultures did not change the general pattern of thalamocortical axonal ingrowth or the average length of collateral branches of these axons. However, in the presence of tetrodotoxin, axons were more highly branched, with an increased number of varicosities as compared to untreated cocultures. This pattern of axonal growth and branching may reflect the activity-dependent fine-tuning and trimming of collaterals that occur as thalamic afferents begin to refine their cortical territory. Our observations in thalamocortical cocultures are consistent with the view that neuronal activity modulates the pattern of axonal growth and development.

Adenovirus-mediated gene transfer into dissociated and explant cultures of rat hippocampal neurons.

Genetic manipulation offers great potential for studying the molecular and cellular processes which control or regulate the complex developmental properties of neurons. Gene transfer into neurons, however, is notoriously difficult. In this study we have used a replication-defective adenovirus (Adv/RSV beta gal), expressing beta-galactosidase (beta-gal) as a reporter gene, to infect dissociated cultures of rat hippocampal neurons and hippocampal slice cultures. Because future studies will require either long-term (e.g., developmental) or short-term (e.g., electrophysiological) expression of recombinant genes in neuronal cultures, we have optimized infection conditions for each situation. The Adv/RSV beta gal construct infects neurons and glial cells equally well, with no apparent alterations in cellular morphology. In slice cultures, the same efficiency and temporal control of beta-gal expression following Adv/RSV beta gal infection was achieved. Focal application of the adenoviruses, by microinjection, permitted infection of discrete subregions within the hippocampal explants. Whole cell recordings of dissociated hippocampal neurons and field recordings from the explant cultures, infected with Adv/RSV beta gal at low multiplicities of infection, indicated no significant alteration in the electrophysiological profiles of neurons in these cultures. The results demonstrate the utility of adenoviruses as gene transfer vectors for primary cultures of neurons. Adenovirus-mediated gene transfer into slice cultures also provides an opportunity to study development or plasticity in an environment where the circuitry and cytoarchitecture of the tissue are preserved and the areas of genetic manipulation can be spatially isolated.

Adenovirus-mediated expression of a reporter gene in thalamocortical cocultures.

Organotypic cocultures of thalamic and cortical explants have recently been used to study the development of the thalamocortical axonal network in the mammalian neocortex. To explore the possibility of genetically manipulating organotypic explants, rat thalamocortical (TC) cocultures were infected with the recombinant adenovirus, Adv/RSV beta gal. Infection of the cortical explants resulted in long-term expression (2 weeks) of the reporter gene (beta-galactosidase) with no significant alterations to the structural integrity of the explants. By micro-injecting the adenoviruses into cortical explants a significant degree of spatial control over reporter gene expression was obtained. DiI-labeled axonal projections from thalamic explants into infected (n = 116) and control cortical (n = 120) explants were also analyzed. There was no significant difference in the extent or degree of TC ingrowth into infected or control cortical explants. Thalamic explants were also efficiently infected with the Adv/RSV beta gal virus. While the pattern and extent of TC ingrowth from infected thalamic explants was similar to controls, the percentage of viable, infected thalamic explants was decreased. These experiments were necessary precursors for future studies using recombinant adenoviruses and organotypic cocultures. Genetic manipulation of these cocultures should enable the dissection of proteins involved in the development of axonal networks in the mammalian neocortex, using a system amenable to direct manipulation and observation.

Effects of vasopressin and aldosterone on the lateral mobility of epithelial Na+ channels in A6 renal epithelial cells.

We have previously demonstrated that apical Na+ channels in A6 renal epithelial cells are associated with spectrin-based membrane cytoskeleton proteins and that the lateral mobility of these channels, as determined by fluorescence photobleach recovery (FPR) analysis, is severely restricted by this association (Smith et al., 1991. Proc. Natl. Acad. Sci. USA 88:6971-6975). Recent data indicate that the actin component of the cytoskeleton may play a role in modulating Na+ channel activity (Cantiello et al., 1991. Am. J. Physiol. 261:C882-C888); however, it is unknown if the Na+ channel's linkage to the spectrin-based membrane cytoskeleton is also involved in regulating channel activity. In this study, we have used FPR to examine if the linkage of the Na+ channels to the membrane cytoskeleton is a site for modulation of Na+ channel activity in filter grown A6 cells by vasopressin and aldosterone. We hypothesized that if the linkage of the Na+ channels to the membrane cytoskeleton is a site for regulation of Na+ channel activity by vasopressin and aldosterone, then hormone-mediated changes in either the membrane cytoskeleton or the affinity of the Na+ channel for the membrane cytoskeleton, should be reflected in changes in the lateral mobility and/or mobile fraction of Na+ channels on the cell surface. FPR revealed that although the rates of lateral mobility were not affected, there was a twofold increase in mobility fraction (f) of apical Na+ channels in aldosterone-treated (16 hr) monolayers (f = 32.31 +/- 5.42%) when compared to control (unstimulated) (f = 14.2 +/- 0.77%) and vasopressin-treated (20 min) (f = 12.7 +/- 2.4%) monolayers. The twofold increase in mobile fraction of Na+ channels corresponds to the average increase in Na+ transport in response to aldosterone in A6 cells. The aldosterone-induced increase in Na+ transport and mobile fraction can be inhibited by the methylation inhibitor, 3-deazaadenosine, consistent with the hypothesis that a methylation event is involved in aldosterone induced upregulation of Na+ transport. We propose that the membrane cytoskeleton is involved in the aldosterone-mediated activation of epithelial Na+ channels.

GABAA/benzodiazepine receptors in acutely isolated hippocampal astrocytes.

The properties of GABA receptor-mediated responses were examined in noncultured astrocytes, acutely isolated from the mature rat hippocampus. Whole-cell patch clamping revealed a GABA-activated Cl- conductance that was mimicked by the GABAA receptor agonist muscimol and depressed by the GABAA antagonists bicuculline and picrotoxin. The GABAA-activated currents were potentiated by the barbiturate pentobarbital and the benzodiazepine diazepam. The benzodiazepine inverse agonist DMCM either enhanced or depressed the astrocytic GABAA-mediated responses, suggesting receptor heterogeneity with respect to pharmacologic profiles. In addition, GABA evoked an increase in [Ca2+]n measured by indo-1 fluorometry, which was depressed in the presence of verapamil or picrotoxin. A GABAA-induced depolarization, therefore, causes Ca2+ influx through voltage-gated Ca2+ channels. The expression and subcellular localization of GABAA receptors and its subunits were examined using immunohistochemical and fluorescent benzodiazepine binding techniques. Polyclonal antisera raised against the GABAA/benzodiazepine receptor, which recognizes multiple subunit isoforms, labeled receptors on the astrocytic cell body and most large processes. In contrast, antisera generated against either alpha 1 or beta 1 subunit peptides revealed immunoreactivity predominantly on a subset of processes. To determine the subcellular distribution of membrane-bound receptors, a fluorescent benzodiazepine derivative was superfused over live astrocytes and visualized with laser-scanning confocal microscopy. Specific fluorescence was distributed in discrete clusters on the cell soma and a subset of distal processes. Collectively, these data support the view that astrocytes, like neurons, express GABAA receptors and target subunit isoforms to distinct cellular localizations. Astrocytic GABAA receptors may be involved in both [Cl-]o and [pH]o homeostasis, and a GABA-evoked increase in [Ca2+]i could serve as a signal between GABAergic neurons and astrocytes.

Distinct temporal patterns of expression of sodium channel-like immunoreactivity during the prenatal development of the monkey and cat retina.

Polyclonal and monoclonal antibodies prepared against the alpha-subunit of the voltage-gated sodium channel (alpha NaCh) were used to examine the distribution of sodium channel-like immunoreactivity during the prenatal development of the cat and rhesus monkey (Macaca mulatta) retina. At all prenatal ages studied, beginning on embryonic day 29 (E29) in the cat and E52 in the monkey, both antibodies labelled optic axons. With the polyclonal antibodies, the appearance of positive cells largely mirrored the onset of their morphological maturation. Immunoreactivity appeared first in the somata of ganglion cells, and subsequently the inner plexiform layer could be distinguished by its intense immunolabelling. A few weeks later horizontal cells displayed immunolabelling that extended to their dendrites in the developing outer plexiform layer. This was followed by immunoreactive cones, with bipolar cells labelled only postnatally. By contrast, with the monoclonal antibody some cells were found to be immunoreactive while their somata were still in the ventricular layer (E33 in cat and E52 in monkey). Many of these cells appeared to migrate to the outer portion of the prospective inner nuclear layer, where they gradually acquired the morphological appearance of bipolar cells. Transient expression of immunolabelling with monoclonal sodium channel antibody was found in the cones of the cat and cones and rods of the monkey. These results indicate that different types of alpha NaCh-like proteins are expressed in the mammalian retina at distinct developmental periods. Their presence at very early stages during development suggests that these proteins could play a specific role in the commitment and/or differentiation of specific retinal cell types.

Tracking movements of lipids and Thy1 molecules in the plasmalemma of living fibroblasts by fluorescence video microscopy with nanometer scale precision.

The lateral diffusion of 100 nm fluorescent latex microspheres (FS) bound to either N-biotinyl-phosphatidyl-ethanolamine or the glycosylphosphatidylinositol-linked protein Thy1 were monitored in the plasmalemma of primary rat fibroblasts by single particle tracking of FS centroids from digital fluorescence micrographs. A silicon intensified target camera was found to be superior to slow scan cooled CCD and intensified interline transfer CCD cameras for monitoring lateral diffusion of rapidly moving FS with nanometer level precision. To estimate the maximum tracking precision, a 4 sec-sequence comprising 120 images of FS fixed to a cover glass was obtained. The mean distance of the centroids from the origin was 7.5 +/- 0.4 nm, and no centroids were beyond 16 nm from the origin. The SIT camera was then used to track FS attached to lipids and Thy1 molecules on the surface of fibroblasts. The lateral diffusion of lipid-bound FS was unconstrained, and the ensemble averaged diffusion coefficient was 0.80 x 10(-9) cm2/sec. Thy1-bound FS existed in two mobility populations, both of which demonstrated constrained mobility. The rapidly moving population, comprising 61% of the total, had an ensemble diffusion coefficient of 6.1 x 10(-10) cm2/sec, and appeared to be restricted to domains with a mean length of about 700 nm. The slowly moving population, comprising about 39% of the total, had a diffusion coefficient of 5.7 x 10(-12) cm2/sec. These results demonstrate that nanovid can be extended to the realm of fluorescence microscopy and support previous studies indicating that while the lateral mobilities of at least some lipids are not constrained to small domains by barriers to lateral diffusion in the fibroblast plasmalemma, a peripheral membrane protein which is bound only by a lipid anchor can be prevented from diffusing freely.

Voltage-dependent sodium channel alpha subunit immunoreactivity is expressed by distinct cell types of the cat and monkey retina.

Polyclonal (7493 and 7317) and monoclonal (mAb3) antibodies, generated to the alpha subunit of the voltage-gated sodium channel (alpha NaCh), were employed to assess the cell types containing alpha NaCh-like immunoreactivity in the mature cat and monkey retina. Immunoblot analyses of retinal proteins in the cat revealed that the polyclonal and monoclonal antibodies we employed labeled a band in the 260-kDa region which corresponds to the molecular mass of the alpha subunit of the NaCh. In both the cat and monkey, these antibodies immunolabeled several distinct types of retinal cells. With the polyclonal antibodies immunoreactivity was observed in ganglion cells and their intraretinal axons, in horizontal cells, and unexpectedly, in cones. In addition, in both species, a limited number of heavily labeled profiles, presumed to be bipolar cells, were seen in the inner nuclear layer. In cat and monkey the monoclonal antibody labeled axons in the fiber layer, ganglion cell somata, and a continuous band of immunoreactive cell bodies (presumed bipolar cells) situated in the outer half of the inner nuclear layer. By immunolabeling isolated cells dissociated from the cat retina, it was possible to demonstrate unequivocally that a population of bipolar cells was labeled by the monoclonal and the polyclonal antibodies we employed. The differences in the labeling observed with the monoclonal antibody as compared to the polyclonal antibodies were interpreted as reflecting the presence of different alpha-subunit subtypes in the mammalian retina. Collectively, our findings suggest that alpha NaCh-like proteins are expressed by a more diverse population of retinal cells than expected on the basis of previous physiological and immunohistochemical studies.

N-Methyl-D-aspartate receptors are clustered and immobilized on dendrites of living cortical neurons.

The response of nerve cells to synaptic inputs and the propagation of this activation is critically dependent on the cell-surface distribution of ion channels. In the hippocampus, Ca2+ influx through N-methyl-D-aspartate receptors (NMDAR) and/or voltage-dependent calcium channels on dendrites is thought to be critically involved in long-term potentiation, neurite outgrowth, epileptogenesis, synaptogenesis, and cell death. We report that conantokin-G (CntxG), a peptide from Conus geographus venom, competitively blocked with high affinity and specificity NMDAR-mediated currents in hippocampal neurons and is a reliable probe for exploring NMDAR distribution. Fluorescent derivatives of CntxG were prepared and used to directly determine NMDAR distribution on living hippocampal neurons by digital imaging and confocal fluorescence microscopy. In hippocampal slices, the CA1 dendritic subfield was strongly labeled by CntxG, whereas the CA3 mossy fiber region was not. On CA1 hippocampal neurons in culture, dendritic CntkG-sensitive NMDAR were clustered at sites of synaptic contacts, whereas somatic NMDAR were distributed diffusely and in patches. NMDAR distribution differed from the distribution of voltage-dependent calcium channels. A significant fraction of labeled NMDAR on somata and dendrites was found to be highly mobile: rates were consistent with the possible rapid recruitment of NMDAR to specific synaptic locations. The localization of NMDAR and modulation of this distribution demonstrated here may have important implications for the events that underlie neuronal processing and synaptic remodeling during associative synaptic modification.

Immunocytochemical study of GABAA receptors in the cat visual cortex.

The laminar distribution and morphological structures associated with GABAA receptor immunoreactivity in the cat visual cortex were studied by using two different polyclonal antibodies directed either against the purified GABAA receptor protein (antibody "967") or against a specific domain of the beta 1-subunit of the GABAA receptor (antibody "Q"). Immunoblots of cat visual cortex tissue with these antibodies revealed that antibody "Q" recognizes only one subunit, namely the beta 1-subunit of the GABAA receptor, and that antibody "967" recognizes three subunits. Both antibodies produced very similar staining patterns, indicating that the beta 1-subunit may be an essential component of the GABAA receptor in the cat visual cortex. The typical staining pattern showed a clear membrane structure around neuronal somata. Using cell body shape criteria, immunopositive neurons included both pyramidal cells in cortical layers II, III, and V, and nonpyramidal cells in all cortical layers. Immunopositive neurons were uniformly distributed in layers II to VI, whereas the density of immunopositive cells in layer I was lower. Some immunopositive neurons were also found in the white matter underlying the visual cortex. In gray matter, immunopositive structures also included dendrites, especially the proximal dendrites, and axon initial segments of pyramidal neurons. The immunopositive processes usually ran vertically toward the pial surface. Some astrocytes were also immunostained. They were localized in layer I and in the white matter. The overall pattern of immunostaining was similar in areas 17, 18, and 19.

Clustering and mobility of voltage-dependent sodium channels during myelination.

In myelinated axons, voltage-dependent sodium channels are segregated at high density at nodes of Ranvier (Rosenbluth, 1976; Waxman and Quick, 1978; Black et al., 1990; Elmer et al., 1990), a distribution that is critical for the saltatory conduction of action potentials (Huxley and Stampfli, 1949). The factors that specifically control the organization and immobilization of sodium channels at nodes are unknown. Recently we have reported that segregation of sodium channels on axons is highly dependent on interactions with active Schwann cells and that continuing axon-glial interactions are necessary to maintain sodium channel distribution during differentiation of myelinated nerve (Joe and Angelides, 1992). The specific recruitment of sodium channels at these early stages of myelination and the conspicuous absence of other axon membrane components suggest that the factors governing sodium channel cluster formation show molecular specificity. However, it is not clear whether these clustered sodium channels originate from a redistribution of preexisting diffusely distributed sodium channels. To determine how Schwann cells might regulate sodium channel distribution during myelination we have examined the lateral mobility of fluorescently labeled sodium channels at defined stages of myelination by fluorescence photobleach recovery using tetramethylrhodamine (TmRhd)-labeled Tityus gamma, a sodium channel-specific fluorescent toxin. First, to test whether Schwann cells, in addition to modulating sodium channel distribution, affect the mobility of sodium channels, we cultured dorsal root ganglion neurons in the presence or absence of Schwann cells and monitored sodium channel mobility on cell bodies, axon hillocks, and axons. Even in the absence of Schwann cells, approximately 80% of the sodium channels were immobile on the time scale of the fluorescence photobleach recovery measurement (DL < or equal to 10(-12) cm2/sec), although the remaining fraction of channels are mobile with diffusion coefficients of 5-13 x 10(-11) cm2/sec. Most importantly, in contrast to the effects of Schwann cells on altering the distribution of sodium channels, we found that Schwann cells did not alter the rate of lateral mobility or the mobile fraction of axonal sodium channels. Therefore, although sodium channel distribution depends on Schwann cell contact, immobilization of sodium channels is independent of Schwann cell contact. These effects appear to be specific for sodium channels because 45% of the succinyl concanavalin-A receptors on the axon are mobile, a fraction that decreases to 25% in the presence of Schwann cells later in development. To determine how sodium channels might be immobilized other than by Schwann cell contact, TmRhd-Tityus gamma-labeled dorsal root neurons were treated with 0.5% Triton X-100.(ABSTRACT TRUNCATED AT 400 WORDS)

Assembly of GABAA receptor subunits determines sorting and localization in polarized cells.

The GABAA receptor, the principal inhibitory receptor in the CNS, is distributed on cell bodies, dendrites, and in some cells at axon hillocks and presynaptic terminals. The dendritic distribution is crucial for shunting of excitatory synaptic inputs. Molecular cloning has revealed that the GABAA receptor can be formed by a diverse set of subunits and by separately encoded subunit isoforms, the expression of each of which differs in distinct areas of the central nervous system and during development. Why different genes exist to encode these isoforms is not clear, but may be linked to functional differences. Here we show that assembly of specific isoforms also codes for sorting and localization of the receptor complex. Confocal microscopy and immunoblot analysis of epithelial cells transfected with the complementary DNAs encoding the alpha 1 and beta 1 GABAA receptor subunits and probed with subunit isoform-specific antibodies show that the alpha 1 subunit is targeted to the basolateral surface, and that the beta 1 subunit is sorted to the apical membrane. In cells where alpha 1 and beta 1 isoforms are co-expressed, assembly of the beta 1 with the alpha 1 subunit isoform re-routes the alpha 1 subunit to the apical surface. The ability to assemble complexes of different isoform composition and to target these to specific regions of the cell surface would enable neurons to modulate GABAA receptor distribution and possibly alter the composition of its synapses in response to transcriptional levels of specific subunit isoforms.

Gastric parietal cell H(+)-K(+)-ATPase microsomes are associated with isoforms of ankyrin and spectrin.

Stimulation of HCl secretion by gastric parietal cells requires the fusion of cytoplasmic H(+)-K(+)-ATPase-bearing tubulovesicles with the apical membrane. This insertion of membrane results in a dramatic increase in apical surface area through the formation of microvilli. To elucidate the elements that may stabilize the newly inserted H(+)-K(+)-ATPase within the apical membrane, we searched for specific cytoskeletal proteins associating with the gastric enzyme. We document by immunoblot analysis that ankyrin, spectrin, and actin copurify with H(+)-K(+)-ATPase microsomes prepared from gastric parietal cells. Coprecipitation of 125I-labeled native erythrocyte ankyrin with the H(+)-K(+)-ATPase from gastric microsomes using anti-H(+)-K(+)-ATPase antibodies suggests that ankyrin associates with the H(+)-K(+)-ATPase. Indirect immunofluorescence and confocal microscopy show that ankyrin and H(+)-K(+)-ATPase cosegregate within resting and secreting parietal cells. Taken together, these data suggest that the association of the gastric H(+)-K(+)-ATPase with spectrin and actin is mediated by ankyrin and that this interaction contributes to the maintenance of the polarized distribution of the enzyme to the apical domain of gastric parietal cells during acid secretion.

Effects of insulin and insulin-like growth factors on neurofilament mRNA and tubulin mRNA content in human neuroblastoma SH-SY5Y cells.

Insulin-like growth factors (IGFs) are implicated in the development of the vertebrate neural circuitry, and increase neurite growth in vitro and in vivo. The construction of the cytoskeleton is necessary for growth of axons and dendrites, and the neurofilament (NF) 68 kDa and 170 kDa proteins assemble to help form major fibrillar elements of the neurite cytoskeleton. We report that physiological concentrations of insulin, IGF-I or IGF-II increased the contents of 68 kDa NF, 170 kDa NF, alpha-tubulin, and beta-tubulin mRNAs, relative to total RNA, in cultured human neuroblastoma SH-SY5Y cells. In contrast, the relative contents of histone 3.3 mRNA, and poly(A)+ RNA were not increased. Ligand concentrations which increased NF mRNAs were very similar to those which increased neurite outgrowth. Although each gene was evidently independently regulated, the 68 kDa NF, 170 kDa NF, alpha-tubulin, and beta-tubulin mRNAs were nevertheless all transiently elevated over approximately the same time interval in response to insulin. These data, when considered together with studies by others with nerve growth factor, show that the 68 kDa and 170 kDa NF mRNAs are elevated in a biochemical pathway activated in common during neurite outgrowth directed by insulin, IGF-I, IGF-II, and nerve growth factor.

Mapping the binding site on ankyrin for the voltage-dependent sodium channel from brain.

Erythrocyte ankyrin is a member of a family of proteins that mediate the linkage between membrane proteins and the underlying spectrin-actin-based cytoskeleton. Ankyrin has been shown to interact with a variety of integral membrane proteins such as the anion exchanger, the Na+K(+)-ATPase, and the voltage-dependent sodium channel (NaCh) in brain. To understand how ankyrin interacts with these proteins and maintains its specificity and high affinity for the voltage-dependent NaCh, we have mapped the binding site on ankyrin for the NaCh by examining the binding of purified ankyrin subfragments, prepared by proteolytic cleavage, to the purified rat brain NaCh incorporated into liposomes. 125I-Labeled ankyrin and the radiolabeled 89- and 43-kDa fragments of ankyrin bind to the NaCh with high affinities and with Kd values of 34, 22, and 63 nM, respectively, and have stoichiometries of approximately 1 mol/mol NaCh. The 72-kDa spectrin binding domain is inactive and does not bind to the NaCh. Dissection of ankyrin reveals that the 43-kDa domain retains all the binding properties of native ankyrin to the NaCh. Analysis of the primary structure reveals that the NaCh binding site is confined to a domain of ankyrin consisting entirely of the 11 terminal 33-amino acid repeats and is distinct from the ankyrin domains that interact with spectrin and the Na+K(+)-ATPase.

GABAA receptor immunoreactivity in the white matter.

Using immunohistochemical methods with polyclonal antibodies directed against a specific sequence of the beta 1-subunit of the GABAA receptor, we found strong immunoreactivity in the white matter of cat brain. The immunopositive products were present primarily on processes of glial cells, especially astrocytes. Immunoreactivity appeared also on the cell bodies of astrocytes and on the cytoplasmic membranes of neurons. The abundant immunostaining in the white matter suggests that (1) GABAA receptors are present on glial cells in vivo, (2) GABAA receptors may be localized on non-synaptic membranes in the white matter and (3) activation of GABAA receptors may have some trophic effects on preservation of the structure and functional properties of the white matter.

Amiloride-sensitive sodium channel is linked to the cytoskeleton in renal epithelial cells.

Amiloride-sensitive sodium channels are localized to the microvillar domain of apical membranes in sodium-transporting renal epithelial cells. To elucidate the elements that maintain sodium channel distribution at the apical membrane, we searched for specific proteins associating with the channel. Triton X-100 extraction of A6 epithelial cells reveals that sodium channels are associated with detergent-insoluble and assembled cytoskeleton. Indirect immunofluorescence and confocal microscopy show that sodium channels are segregated to the apical microvillar membrane and colocalize with ankyrin, fodrin, and actin. We document by immunoblot analysis that ankyrin and fodrin remain associated with sodium channels after isolation and purification from bovine renal papillae. 125I-labeled ankyrine can be precipitated by anti-sodium-channel antibodies only in the presence of purified bovine sodium-channel complex. Direct binding of 125I-labeled ankyrin shows ankyrin binds to the 150-kDa subunit of the channel. Fluorescence photobleach lateral-diffusion measurements indicate sodium channels are severely restricted in their lateral mobility. We conclude that ankyrin links the amiloride-sensitive sodium channel to the underlying cytoskeleton and this association may sequester sodium channels at apical microvilli and maintain their polarized distribution in renal epithelial cells.

Distribution of Ca2+ channels on frog motor nerve terminals revealed by fluorescent omega-conotoxin.

Tetramethylrhodamine-conjugated omega-conotoxin was used as a fluorescent stain (Jones et al., 1989) to determine the spatial distribution of voltage-gated Ca2+ channels along frog motor nerve terminals. Like native omega-conotoxin, the fluorescent toxin blocked neuromuscular transmission irreversibly. The fluorescent staining was confined to the neuromuscular junction and consisted of a series of narrow bands (in face views) or dots (in side views) approximately 1 micron apart. This characteristic staining pattern was prevented by pretreatment with omega-conotoxin and by prior denervation for 5-7 d. Combined fluorescence and phase-contrast optics indicated that the stain was on the synaptic rather than the nonsynaptic side of the nerve terminal. The bands and dots of stain proved to be in spatial register with the postsynaptic junctional folds, as revealed by combined staining of ACh receptors. It is concluded that the voltage-gated Ca2+ channels on frog motor nerve terminals are concentrated at active zones. The findings are consistent with the suggestion (Heuser et al., 1974; Pumplin et al., 1981) that the large intramembraneous particles seen at freeze-fractured active zones are voltage-gated Ca2+ channels.