Sodium channel Na(v)1.6 is localized at nodes of ranvier, dendrites, and synapses.

Voltage-gated sodium channels perform critical roles for electrical signaling in the nervous system by generating action potentials in axons and in dendrites. At least 10 genes encode sodium channels in mammals, but specific physiological roles that distinguish each of these isoforms are not known. One possibility is that each isoform is expressed in a restricted set of cell types or is targeted to a specific domain of a neuron or muscle cell. Using affinity-purified isoform-specific antibodies, we find that Na(v)1.6 is highly concentrated at nodes of Ranvier of both sensory and motor axons in the peripheral nervous system and at nodes in the central nervous system. The specificity of this antibody was also demonstrated with the Na(v)1.6-deficient mouse mutant strain med, whose nodes were negative for Na(v)1.6 immunostaining. Both the intensity of labeling and the failure of other isoform-specific antibodies to label nodes suggest that Na(v)1.6 is the predominant channel type in this structure. In the central nervous system, Na(v)1.6 is localized in unmyelinated axons in the retina and cerebellum and is strongly expressed in dendrites of cortical pyramidal cells and cerebellar Purkinje cells. Ultrastructural studies indicate that labeling in dendrites is both intracellular and on dendritic shaft membranes. Remarkably, Na(v)1.6 labeling was observed at both presynaptic and postsynaptic membranes in the cortex and cerebellum. Thus, a single sodium channel isoform is targeted to different neuronal domains and can influence both axonal conduction and synaptic responses.

Immunolocalization of sodium channel isoform NaCh6 in the nervous system.

Sodium channel 6 (NaCh6) is the alpha-subunit of a voltage-gated sodium channel expressed in the rat nervous system. The mRNA for this isoform has been shown to be expressed in both neuronal and glial cells by in situ hybridization. To examine localization of NaCh6 protein, polyclonal antibodies specific for NaCh6 were generated against peptides from two cytoplasmic domains and a fusion protein from an extracellular domain. Affinity-purified antibodies were used to localize NaCh6 in the brain, spinal cord, peripheral nervous system, and neuromuscular junction. There was widespread labeling of neurons in the brain and spinal cord. NaCh6 was present in both sensory and motor pathways. Radial glial cells in the cerebellum were intensely labeled for both GFAP and NaCh6. At the subcellular level, NaCh6 is found in axons, dendrites, and the cell body. Motor neurons and primary sensory neurons in dorsal root ganglia had strong cytoplasmic and axonal staining. Nodes of Ranvier in peripheral nerve and in the spinal cord were also intensely labeled. Motor neuron axons near the neuromuscular junction were labeled up to, but not including, terminal boutons. Dendrites of pyramidal cells in the cortex, hippocampus, and cerebellum were labeled. NaCh6 is the first NaCh subtype to be localized either at the node of Ranvier or to a dendrite. We conclude that NaCh6 is widely distributed in the central and peripheral nervous systems and is likely to be important for the electrical properties of the axon and dendrite.

Developmental and regional expression of sodium channel isoform NaCh6 in the rat central nervous system.

The sodium channel isoform NaCh6 is abundant in the adult rat brain and is expressed in both neurons and glia (Schaller et al. [1995] J. Neurosci. 15:3231-3242; Krzemien et al. [2000] J. Comp. Neurol. 20:70-83). With reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunolabeling, NaCh6 expression was investigated in the developing rat brain and spinal cord [embryonic day 15 (E15) through postnatal day 28 (P28)]. The relative abundance of the four major central nervous system NaCh subtypes was quantitated with RT-PCR. In all regions that were investigated (olfactory bulb, cortex, hippocampus, cerebellum, and spinal cord), each subtype had a unique pattern of expression. NaCh6 mRNA and protein were not detected in either brain or spinal cord at E15 and E18 by in situ hybridization and immunohistochemistry. Neurons in the hippocampus, cortex, and olfactory bulb began to express NaCh6 mRNA and protein shortly after birth. The mRNA signal peaked at P7-P14, and protein expression increased as development proceeded. NaCh6 mRNA was detected at P1 in the cerebellum, and a nonuniform distribution of NaCh6 immunoreactivity in both Purkinje cells and granule cells was observed by P7-P14. NaCh6 protein was expressed in granule cells as soon as they left the proliferative phase and began to migrate. Both NaCh6 mRNA and protein were detected in the spinal cord at P1 and were expressed clearly at P7 in motor neurons. The time course of appearance of NaCh6 in postnatal development is consistent with the development of neurologic symptoms in med and jolting mice, which have mutations in the mouse ortholog of NaCh6.

A novel, abundant sodium channel expressed in neurons and glia.

A novel, voltage-gated sodium channel cDNA, designated NaCh6, has been isolated from the rat central and peripheral nervous systems. RNase protection assays showed that NaCh6 is highly expressed in the brain, and NaCh6 mRNA is as abundant or more abundant than the mRNAs for previously identified rat brain sodium channels. In situ hybridization demonstrated that a wide variety of neurons express NaCh6, including motor neurons in the brainstem and spinal cord, cerebellar granule cells, and pyramidal and granule cells of the hippocampus. RT-PCR and/or in situ hybridization showed that astrocytes and Schwann cells express NaCh6. Thus, this sodium channel is broadly distributed throughout the nervous system and is shown to be expressed in both neurons and glial cells.

Expression and distribution of sodium channels in short- and long-term denervated rodent skeletal muscles.

1. Loose-patch voltage-clamp recordings were made from rat and mouse skeletal muscle fibres denervated for up to 6 weeks. Innervated muscles possessed a Na+ current density of 107 +/- 3.3 mA cm-2 in endplate membrane, and 6.3 +/- 0.6 mA cm-2 in extrajunctional membrane. This high concentration of Na+ channels at the endplate was gradually reduced following denervation. After 6 weeks of denervation, the endplate Na+ channel concentration was reduced by 40-50%, and the density of Na+ channels in extrajunctional membrane was increased by about 30%. 2. The tetrodotoxin (TTX)-resistant form of the Na+ channel appeared after 3 days of denervation and comprised approximately 43% of the endplate Na+ channels 5-6 days after denervation. Subsequently, TTX-resistant Na+ channels were reduced in density to approximately 25% of the postjunctional Na+ channels and remained at this level up to 6 weeks after denervation. 3. RNase protection analysis showed that mRNA encoding the TTX-resistant Na+ channel was virtually absent in innervated muscle, rose > 50-fold after 3 days of denervation, then decreased by 95% 6 weeks after denervation. The density of TTX-resistant Na+ channels correlated qualitatively with changes in mRNA levels. 4. These results suggest that the density of Na+ channels at neuromuscular junctions is maintained by two mechanisms, one influenced by the nerve terminal and the other independent of innervation.

Muscle sodium channel inactivation defect in paramyotonia congenita with the thr1313met mutation.

Mutations of the skeletal muscle sodium (Na) channel have been reported in families with paramyotonia congenita (PC), an autosomal dominant disorder with cold and/or exercise induced stiffness and myotonia. Functional consequences of specific Na channel mutations responsible for PC have not been described. Patch clamp recording of single Na channels were made in cultured myotubes at 22 and 34 degrees C from a PC patient with the thr1313met mutation. Cell-attached and outside-out recordings of mutant PC channels contained long duration and late openings. The mean open time was increased and the ensemble average showed a prolonged inward Na current. This membrane depolarization could cause repetitive action potentials and the clinical syndrome.

Aggregation of sodium channels during development and maturation of the neuromuscular junction.

The voltage-activated Na channel (NaCh) is an integral membrane protein that is enriched at the neuromuscular end plate. Using loose-patch voltage-clamp and immunofluorescence, we have found that the aggregation of NaChs occurs late, during maturation of the neuromuscular junction. A decline in expression of embryonic NaCh mRNA and increase in adult NaCh mRNA precedes the onset of aggregation, and the appearance of functional adult NaChs coincides with NaCh aggregation. We tested the possibility that only the adult NaCh subtype could aggregate during development and found that both the embryonic and adult isoforms become concentrated at the synapse. The NaCh is the first postsynaptic membrane protein shown to become clustered postnatally, and the mechanism producing this aggregation appears to be different from the process producing aggregation of other synaptic proteins.

Alternatively spliced sodium channel transcripts in brain and muscle.

Sodium (Na) channel cDNAs were synthesized from RNA isolated from rat brain, cardiac muscle, and skeletal muscle. Partial cDNAs coding for the largest cytoplasmic loop of the Na channel were amplified with PCR. Sequence analysis of these cDNAs revealed that Na channel cDNAs originally described as brain genes were also expressed in both cardiac and skeletal muscle. Some of these cDNAs were isoforms that differed by insertions or deletions and can be explained by alternative choices of a 5' splice site. Southern blot analysis of genomic DNA confirmed the presence of introns in this region of the gene. Transcripts of multiple isoforms were detected with RNase protection in brain, heart, and skeletal muscle. Several conclusions can be drawn from the data. (1) Some rat sodium channel genes are transcribed in all excitable tissues studied here: brain, cardiac muscle, and skeletal muscle. (2) Each of these three tissues expresses multiple sodium channel genes. (3) Alternative splicing of sodium channel transcripts occurs in these tissues. (4) Expression of multiple genes and alternative splicing of the transcripts is responsible for at least seven different sodium channel mRNAs in skeletal muscle.

Translocation of cAMP-dependent protein kinase to the nucleus during development of Dictyostelium discoideum.

The distribution of the catalytic and regulatory subunits of the cAMP-dependent protein kinase between cytoplasm and nucleus was determined during the development of Dictyostelium discoideum. In vegetative amoebae approximately 2% of the subunits were in the nucleus. During development there was an approximately 5-fold increase in total soluble cAMP-dependent protein kinase and a 15- to 30-fold increase of enzyme in the nuclear fraction. There was a reverse translocation from nucleus to cytoplasm, when Tipped Aggregates were disrupted and the resultant amoebae incubated in single-cell suspension. The addition of cAMP to these single-cell suspensions brought about the reentry of the subunits into the nucleus. The findings are discussed in relation to the potential role of the cAMP-dependent protein kinase in the regulation of mRNA and protein synthesis.

Differential cellular distribution of cAMP-dependent protein kinase during development of Dictyostelium discoideum.

It was shown previously by us that cAMP-dependent protein kinase activity in the cellular slime mold Dictyostelium discoideum increased during the early stages of development. Results from other laboratories showed that during the subsequent stage of cell differentiation and positioning, the accumulation of a number of prespore mRNAs and proteins (but not prestalk mRNAs and proteins) was dependent upon cAMP. The present communication describes the cellular distribution of the cAMP-dependent protein kinase at that stage of development. Pseudoplasmodia were disrupted, and prespore cells were separated from prestalk cells by sedimentation through a Percoll gradient. Prespore cells had approximately 4-5 times as much of both the catalytic and regulatory subunits of the cAMP-dependent protein kinase as did the prestalk cells. That the increase of cAMP-dependent protein kinase during development reflected de novo synthesis of the enzyme in both prespore and prestalk cells was demonstrated on the basis of [(3)H]leucine incorporation into the regulatory subunit. The findings are consistent with a role of the cAMP-dependent protein kinase in mediating the effects of cAMP on the synthesis of prespore-specific mRNAs and proteins at the stage at which cAMP appears to be required for the cell type-specific syntheses.

A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discoideum. II. Developmental regulation.

The cAMP-dependent protein kinase of the cellular slime mold, Dictyostelium discoideum, is developmentally regulated; there is an approximately 4-fold increase in activity during development. The incorporation of [3H]leucine into the enzyme demonstrates that there is de novo synthesis of the cAMP-dependent protein kinase. The activities of the catalytic and regulatory subunits increase in parallel. The maximal rate of increase of cAMP-dependent protein kinase activity precedes "tip" formation, a stage of development characterized by a sharp increase in mRNA complexity. The high level of cAMP-dependent protein kinase activity, attained at this stage of development, persists when aggregates are dispersed and the amoebae are kept in suspension without added cAMP. The synthesis of the developmentally regulated mRNAs under these conditions is dependent on exogenous cAMP. The increase in cAMP-dependent protein kinase activity during development does not require sustained cell-cell contact insofar as it occurs in single cell suspensions of amoebae. Furthermore, the increase does not require exogenous cAMP, although added cAMP stimulates the synthesis of the enzyme to a level higher than that found, when cAMP is not added. These observations support the hypothesis that in D. discoideum cAMP-dependent protein kinase mediates the effects of cAMP on development.