John H. Caldwell

Compact Myelin Dictates the Differential Targeting of Two Sodium Channel Isoforms in the Same Axon

Voltage-dependent sodium channels are uniformly distributed along unmyelinated axons, but are highly concentrated at nodes of Ranvier in myelinated axons. Here, we show that this pattern is associated with differential localization of distinct sodium channel alpha subunits to the unmyelinated and myelinated zones of the same retinal ganglion cell axons. In adult axons, Na(v)1.2 is localized to the unmyelinated zone, whereas Na(v)1.6 is specifically targeted to nodes. During development, Na(v)1.2 is expressed first and becomes clustered at immature nodes of Ranvier, but as myelination proceeds, Na(v)1.6 replaces Na(v)1.2 at nodes. In Shiverer mice, which lack compact myelin, Na(v)1.2 is found throughout adult axons, whereas little Na(v)1.6 is detected. Together, these data show that sodium channel isoforms are differentially targeted to distinct domains of the same axon in a process associated with formation of compact myelin.

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.

APP and APLP2 are essential at PNS and CNS synapses for transmission, spatial learning and LTP

Despite its key role in Alzheimer pathogenesis, the physiological function(s) of the amyloid precursor protein (APP) and its proteolytic fragments are still poorly understood. Previously, we generated APPsα knock‐in (KI) mice expressing solely the secreted ectodomain APPsα. Here, we generated double mutants (APPsα‐DM) by crossing APPsα‐KI mice onto an APLP2‐deficient background and show that APPsα rescues the postnatal lethality of the majority of APP/APLP2 double knockout mice. Surviving APPsα‐DM mice exhibited impaired neuromuscular transmission, with reductions in quantal content, readily releasable pool, and ability to sustain vesicle release that resulted in muscular weakness. We show that these defects may be due to loss of an APP/Mint2/Munc18 complex. Moreover, APPsα‐DM muscle showed fragmented post‐synaptic specializations, suggesting impaired postnatal synaptic maturation and/or maintenance. Despite normal CNS morphology and unaltered basal synaptic transmission, young APPsα‐DM mice already showed pronounced hippocampal dysfunction, impaired spatial learning and a deficit in LTP that could be rescued by GABAA receptor inhibition. Collectively, our data show that APLP2 and APP are synergistically required to mediate neuromuscular transmission, spatial learning and synaptic plasticity.

SCN5A Mutations Associate With Arrhythmic Dilated Cardiomyopathy and Commonly Localize to the Voltage-Sensing Mechanism.

OBJECTIVES The aim of this study was to discern the role of the cardiac voltage-gated sodium ion channel SCN5A in the etiology of dilated cardiomyopathy (DCM). BACKGROUND Dilated cardiomyopathy associates with mutations in the SCN5A gene, but the frequency, phenotype, and causative nature of these associations remain the focus of ongoing investigation. METHODS Since 1991, DCM probands and family members have been enrolled in the Familial Cardiomyopathy Registry and extensively evaluated by clinical phenotype. Genomic deoxyribonucleic acid samples from 338 individuals among 289 DCM families were obtained and screened for SCN5A mutations by denaturing high-performance liquid chromatography and sequence analysis. RESULTS We identified 5 missense SCN5A mutations among our DCM families, including novel mutations E446K, F1520L, and V1279I, as well as previously reported mutations D1275N and R222Q. Of 15 SCN5A mutation carriers in our study, 14 (93%) manifested arrhythmia: supraventricular arrhythmia (13 of 15), including sick sinus syndrome (5 of 15) and atrial fibrillation (9 of 15), ventricular tachycardia (5 of 15), and conduction disease (9 of 15). CONCLUSIONS Mutations in SCN5A were detected in 1.7% of DCM families. Two-thirds (6 of 9) of all reported DCM mutations in SCN5A localize to the highly conserved homologous S3 and S4 transmembrane segments, suggesting a shared mechanism of disruption of the voltage-sensing mechanism of this channel leading to DCM. Not surprisingly, SCN5A mutation carriers show a strong arrhythmic pattern that has clinical and diagnostic implications.

Immunolocalization of sodium channel isoform NaCh6 in the nervous system

Sodium channel 6 (NaCh6) is the α‐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. J. Comp. Neurol. 420:70–83, 2000.

Carbon Nanotubes Promote Growth and Spontaneous Electrical Activity in Cultured Cardiac Myocytes

Nanoscale manipulations of the extracellular microenvironment are increasingly attracting attention in tissue engineering. Here, combining microscopy, biological, and single-cell electrophysiological methodologies, we demonstrate that neonatal rat ventricular myocytes cultured on substrates of multiwall carbon nanotubes interact with carbon nanotubes by forming tight contacts and show increased viability and proliferation. Furthermore, we observed changes in the electrophysiological properties of cardiomyocytes, suggesting that carbon nanotubes are able to promote cardiomyocyte maturation.

Expression and distribution of voltage-gated sodium channels in the cerebellum.

In order to understand the effects of sodium channels on synaptic signaling and response in the cerebellum, it is essential to know for each class of neuron what sodium channel isoforms are present, and the properties and distribution of each. Sodium channels are heteromultimeric membrane proteins, consisting of a large alpha subunit that forms the pore, and one or more beta subunits. Ten genes encode an alpha subunit in mammals, and of these, four are expressed in the cerebellum: Navl.l, Nav1.2, Nav1.3 and Nav1.6. Three genes encode beta subunits (Naβl–3), and all three are expressed in the cerebellum. However, Nav1.3 and Naβ3 have been found only in the developing cerebellum. All sodium channels recorded in the cerebellum are TTX-sensitive with similar kinetics, making it difficult to identify the isoforms electrically. Thus, most of the expression studies have relied on techniques that allow visualization of sodium channel subtypes at the level of mRNA and protein. In situ hybridization and immunolocalization studies demonstrated that granule cells predominantly express Nav1.2, Nav1.6, Naβ1, and Naβ2. Protein for Nav1.2 and Nav1.6 is localized primarily in granule cell parallel fibers. Purkinje cells express Nav1.1, Nav1.6, Naβl and Naβ2. The somatodendritic localization of Navl.l and Nav1.6 in Purkinje cells suggests that these isoforms are involved in the integration of synaptic input. Deep cerebellar nuclei neurons expressed Nav1.1 and Nav1.6 as well as Naβ1. Bergmann glia expressed Nav1.6, but not granule cell layer astrocytes. Some sodium channel isoforms that are not expressed normally in the adult cerebellum are expressed in animals with mutations or disease. Electrophysiological studies suggest that Nav1.6 is responsible for spontaneous firing and bursting features in Purkinje cells, but the specialized functions of the other subunits in the cerebellum remain unknown.

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. J. Comp. Neurol. 420:84–97, 2000.

Modulation of skeletal and cardiac voltage-gated sodium channels by calmodulin

Calmodulin (CaM) has been shown to modulate different ion channels, including voltage‐gated sodium channels (NaChs). Using the yeast two‐hybrid assay, we found an interaction between CaM and the C‐terminal domains of adult skeletal (NaV1.4) and cardiac (NaV1.5) muscle NaChs. Effects of CaM were studied using sodium channels transiently expressed in CHO cells. Wild type CaM (CaMWT) caused a hyperpolarizing shift in the voltage dependence of activation and inactivation for NaV1.4 and activation for NaV1.5. Intracellular application of CaM caused hyperpolarizing shifts equivalent to those seen with CaMWT coexpression with NaV1.4. Elevated Ca2+ and CaM‐binding peptides caused depolarizing shifts in the inactivation curves seen with CaMWT coexpression with NaV1.4. KN93, a CaM‐kinase II inhibitor, had no effect on NaV1.4, suggesting that CaM acts directly on NaV1.4 and not through activation of CaM‐kinase II. Coexpression of hemi‐mutant CaMs showed that an intact N‐terminal lobe of CaM is required for effects of CaM upon NaV1.4. Mutations in the sodium channel IQ domain disrupted the effects of CaM on NaV1.4: the I1727E mutation completely blocked all calmodulin effects, while the L1736R mutation disrupted the effects of Ca2+–calmodulin on inactivation. Chimeric channels of NaV1.4 and NaV1.5 also indicated that the C‐terminal domain is largely responsible for CaM effects on inactivation. CaM had little effect on NaV1.4 expressed in HEK cells, possibly due to large differences in the endogenous expression of β‐subunits between CHO and HEK cells. These results in heterologous cells suggest that Ca2+ released during muscle contraction rapidly modulates NaCh availability via CaM.

Cell Polarity: Endogenous Ion Currents Precede and Predict Branching in the Water Mold Achyla

The hyphae of the water mold Achyla bisexualis generate electrical currents that enter the growing tips and leave farther back. An inward-moving current also precedes branching and predicts the site of branch emergence; during the branching process, the current at the original tip declines or even reverses transiently without any change in growth rate. The inward current probably acts as an early signal during branch differentiation. The flow of specific ions rather than the flow of electrical charge probably serves to localize growth.