Simon R. Levinson

Neuraminidase treatment modifies the function of electroplax sodium channels in planar lipid bilayers

Sodium channels from several sources are covalently modified by unusually large numbers of negatively charged sialic acid residues. In the present studies, purified electroplax sodium channels were treated with neuraminidase to remove sialic acid residues and then examined for functional changes in planar lipid bilayers. Neuraminidase treatment resulted in a large depolarizing shift in the average potential required for channel activation. Additionally, desialidated channels showed a striking increase in the frequency of reversible transitions to subconductance states. Thus it appears that sialic acid residues play a significant role in the function of sodium channels, possibly through their influence on the local electric field and/or conformational stability of the channel molecule.

The Sodium Channel from Electrophorus electricusa

The purification of the sodium channel from excitable tissues has been a crucial step toward the elucidation of channel mechanism and molecular biology. In turn, this advance required the development of assays for channels during their fractionation from disrupted and solubilized membranes (which obviously could not be based on their normal physiological role as voltage-gated mediators of ion transport into intact cells). Fortunately, such precise and sensitive assays have been achieved through the channel-specific, high affinity binding of tetrodotoxin (TTX) and saxitoxin.”’ In addition, when bound to the channel these toxins prevent their own binding site from denaturing during the lengthy purification procedures employed, allowing a high proportion of channel protein to be purified which retains the ability to bind toxin.3 Thus both TTX and saxitoxin have played a vital role in the study of the molecular biology of the sodium channel. Naturally successful purification of sodium channels also requires a rich tissue source which is readily available and amenable to biochemical isolation techniques. To the naive reader the use of the electric eel Electrophorus electricus as a source of channel material may seem unnecessarily exotic, but in fact these beasts are endowed with amazingly large quantities of sodium channels a t high d e n ~ i t y . ~ As a result, the eel electroplax channel has played a prominent role in channel studies, having now been p~rified,’.~ chemically characterized5 and sequenced,6 and visualized both by electron microscopy’ and immunocytochemical techniques? In addition, the function and pharmacology of the eel channel appear to be very similar to channels from other animals, so that there is reason to suppose that molecular information derived from electroplax channels will be broadly applicable to those from other sources. The success of the electric organ preparation is probably due as much to the richness of the tissue in sodium channels as to the skill (and luck!) of the investigators. A crude fraction of electroplax membranes has about 0.5% sodium channel relative to

THE ROLE OF SODIUM CHANNELS IN CHRONIC PAIN

Here we review recent research into the mechanisms of chronic pain that has focused on neuronal sodium channels, a target of classic analgesic agents. We first discuss evidence that specific sodium channel isoforms are essential for the detection and conduction of normal acutely painful stimuli from nociceptors. We then review findings that show changes in sodium channel expression and localization in chronic inflammation and nerve injury in animal and human tissues. We conclude by discussing the role that myelination plays in organizing and maintaining sodium channel clusters at nodes of Ranvier in normal development and how inflammatory processes or nerve injury alter the characteristics of such clusters. Based on these findings, we suggest that chronic pain may in part result from partial demyelination of axons during chronic injury, which creates aberrant sodium channel clusters that serve as sites of ectopic sensitivity or spontaneous activity. Muscle Nerve 46: 155–165, 2012

Pulpitis increases the proportion of atypical nodes of Ranvier in human dental pulp axons without a change in Nav1.6 sodium channel expression

Studies show a change in sodium channel (NaCh) expression after inflammatory lesions, and this change is implicated in the generation of pain states. We are using the extracted human tooth to study NaCh expression and here examine the expression of the major NaCh isoform located at nodes of Ranvier, Na(v)1.6, in normal and painful samples. Pulpal sections were double-labeled with human-specific Na(v)1.6 antibody and caspr antibody (paranodal protein to identify nodes). Confocal microscopy was used to obtain a z-series of optically-sectioned images of axon bundles surrounded by inflammatory cells in painful samples and of similar regions within the coronal pulp of normal samples. Nodes contained within these images were classified as typical or atypical as based on caspr staining relationships, and NIH ImageJ software was used to quantify the size and immunofluorescence staining intensity of Na(v)1.6 accumulations at these nodal sites. Results show no significant difference in the size or immunofluorescence staining intensity of Na(v)1.6 nodal accumulations located at either typical or atypical nodal sites (heminodes and split nodes) within axons in normal samples when compared to painful samples (n=9/each group). In contrast, there was a highly significant decrease in the proportion of typical nodal sites and an increase in atypical nodal sites in painful samples when compared to normal samples. The unchanged expression of Na(v)1.6 contrasts to our previous finding that showed an increased expression of Na(v)1.7 at both typical and atypical nodal sites within painful samples. Together, these findings suggest there is not a simple replacement of one isoform with another, but rather an increased co-expression of multiple isoforms at both intact and remodeling/demyelinating (atypical) nodal sites within the painful dental pulp. The resultant heterogeneous population of isoforms may produce unique axonal excitability properties that could contribute to spontaneous pain sensations that are common in toothache.

Characterization of Nuclear Neurokinin 3 Receptor Expression in Rat Brain

Ligand-induced translocation of the G-protein-coupled receptor, neurokinin 3 (NK3-R), to the nucleus of hypothalamic neurons was reported using antibodies (ABs) raised against the C-terminal region of NK3-R. The current work was undertaken to substantiate the ability of NK3-R to enter the nucleus and identify which portion of the NK3-R molecule enters the nucleus. ABs directed at epitopes in the N-terminal and second extracellular loop of the rat NK3-R molecule were used to evaluate western blots of whole tissue homogenates and nuclear fractions from multiple brain areas. Specificity of the protein bands recognized by these ABs was demonstrated using Chinese hamster ovary (CHO) cells transfected with rat or human NK3-R. Both ABs prominently recognized a diffuse protein band of ∼56-65 kDa (56 kDa=predicted size) and distinct ∼70-kDa and 95-kDa proteins in homogenates of multiple brain areas. The ∼95-kDa protein recognized by the extracellular loop AB was enriched in nuclear fractions. Recognition of these proteins by ABs directed at different regions of the NK3-R supports their identification as NK3-R. The size differences reflect variable glycosylation and possibly linkage to different cytosolic and nuclear proteins. Recognition of protein bands by both ABs in nuclear fractions is consistent with the full-length NK3-R entering the nucleus. Hypotension increased the density of the ∼95-kDa band in nuclear fractions from the supraoptic nucleus indicating activity-induced nuclear translocation. Since NK3-R is widely distributed in the CNS, the presence of NK3-R in nuclei from multiple brain regions suggests that it may broadly influence CNS gene expression in a ligand-dependent manner.

NaV1.5 sodium channel window currents contribute to spontaneous firing in olfactory sensory neurons

Olfactory sensory neurons (OSNs) fire spontaneously as well as in response to odor; both forms of firing are physiologically important. We studied voltage-gated Na(+) channels in OSNs to assess their role in spontaneous activity. Whole cell patch-clamp recordings from OSNs demonstrated both tetrodotoxin-sensitive and tetrodotoxin-resistant components of Na(+) current. RT-PCR showed mRNAs for five of the nine different Na(+) channel α-subunits in olfactory tissue; only one was tetrodotoxin resistant, the so-called cardiac subtype NaV1.5. Immunohistochemical analysis indicated that NaV1.5 is present in the apical knob of OSN dendrites but not in the axon. The NaV1.5 channels in OSNs exhibited two important features: 1) a half-inactivation potential near -100 mV, well below the resting potential, and 2) a window current centered near the resting potential. The negative half-inactivation potential renders most NaV1.5 channels in OSNs inactivated at the resting potential, while the window current indicates that the minor fraction of noninactivated NaV1.5 channels have a small probability of opening spontaneously at the resting potential. When the tetrodotoxin-sensitive Na(+) channels were blocked by nanomolar tetrodotoxin at the resting potential, spontaneous firing was suppressed as expected. Furthermore, selectively blocking NaV1.5 channels with Zn(2+) in the absence of tetrodotoxin also suppressed spontaneous firing, indicating that NaV1.5 channels are required for spontaneous activity despite resting inactivation. We propose that window currents produced by noninactivated NaV1.5 channels are one source of the generator potentials that trigger spontaneous firing, while the upstroke and propagation of action potentials in OSNs are borne by the tetrodotoxin-sensitive Na(+) channel subtypes.

Monoclonal antibodies raised against post-translational domains of the electroplax sodium channel

SummaryEleven monoclonal antibodies were identified that recognized eel electroplax sodium channels. All the monoclonal antibodies specifically immunostained the mature TTX-sensitive sodium channel (Mr 265,000) on immunoblots. None of the monoclonal antibodies would precipitate the in vitro translated channel core polypeptide in solution. One monoclonal antibody, 3G4, was found to bind to an epitope involving terminal polysialic acids. Extensive digestion of the channel by the exosialidase, neuraminidase, or partial polysialic acid removal bythe endosialidase, endo-N-acetylneuraminidase, destroy the 3G4 epitope, 3G4 is, therefore, a highly selective probe for the post-translationally attached polysialic acids. Except for this monoclonal antibody, the epitopes recognized by the remaining antibodies were highly resistant to extensive N-linked deglycosylation. Thus, the monoclonal antibodies may be directed against unique post-translationally produced domains of the electroplax sodium channel, presumably sugar groups that are abundant on this protein (Miller, J.A., Agnew, W.S., Levinson, S.R. 1983.Biochemistry22:462–470). These monoclonal antibodies should prove useful as tools to study discrete post-translational processing events in sodium channel biosynthesis.