Topology and patch-clamp analysis of the sodium channel in relationship to the anti-lipid a antibody in campylobacteriosis (original) (raw)
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Histology and histopathology, 2008
The aim of this work was to characterize several ionic channels in nervous cells of the suboesophageal visceral, left and right parietal, and left and right pleural brain ganglia complex of the snail Helix aspersa by immunocytochemistry. We have studied the immunostaining reaction for a wide panel of eleven polyclonal antibodies raised against mammal antigens as follows: voltage-gated-Na+ channel; voltage-gated-delayed-rectifier-K+ channel; SK2-small-conductance-Ca2+-dependent-K+ channel apamin sensitive; SK3 potassium channel; charybdotoxin-sensitive voltage-dependent potassium channel; BKCa-maxi-conductance-Ca2+-dependent-K+ channel; hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; G-protein-activated inwardly rectifying potassium channel GIRK2 and voltage-gated-calcium of L, N and P/Q type channels. Our results show positive reaction in neurons, but neither in glia cells nor in processes in the Helix suboesophageal ganglia. Our results suggest the occurren...
Tetanus Toxin Forms Channels in Planar Lipid Bilayers Containing Gangliosides
Biophysical Journal, 1984
Although the agent responsible for the spasticity and convulsions characteristic of human tetanus was identifed at the turn of the century as the neurotoxin produced by the bacterium Clostridium tetani (1), the molecular basis for the action of the toxin at the central nervous system remains obscure (1, 2). Tetanus toxin (TT), a soluble protein of M, -150,000 in its native form, has no enzymatic activity identified thus far. It is known that TT binds specifically to gangliosides with a higher affinity to GDI b and GTIb (3). These sialo sphingolipids are especially abundant in nervous tissues. The question now arises: How is TT binding to gangliosides related to its neurotoxicity? We investigated the effect of the TT-ganglioside interaction in membranes by assaying the formation of transmembrane ionic channels in planar lipid bilayers. Asymmetric lipid bilayers (4) were formed by the apposition of a monolayer of asolectin (soybean phospholipids, AL) and a monolayer of AL supplemented with gangliosides (G). TT was added to one of the aqueous compartments and the current under voltage clamp was recorded. We found that TT forms channels only when added to the compartment in contact with the ganglioside face of the membrane. The TT channel is cation-selective and its residence time in the open state is longer than in the closed state. A preliminary account of this research appeared elsewhere (5).
Journal of Biological Chemistry, 2006
In a recent note to Nature, R. MacKinnon has raised the possibility that potassium channel gating modifiers are able to partition in the phospholipid bilayer of neuronal membranes and that by increasing their partial concentration adjacent to their receptor, they affect channel function with apparent high affinity (Lee and MacKinnon 2004) Nature 430, 232-235). This suggestion was adopted by Smith et al. (Smith, J. J., Alphy, S., Seibert, A. L., and Blumenthal, K. M. (2005) J. Biol. Chem. 280, 11127-11133)
µO-Conotoxins Inhibit NaV Channels by Interfering with their Voltage Sensors in Domain-2
…, 2007
The µO-conotoxins MrVIA and MrVIB are 31-residue peptides from Conus marmoreus, belonging to the O-superfamily of conotoxins with three disulfide bridges. They have attracted attention because they are inhibitors of tetrodotoxin-insensitive voltage-gated sodium channels (Na V 1.8) and could therefore serve as lead structure for novel analgesics. The aim of this study was to elucidate the molecular mechanism by which µO-conotoxins affect Na V channels. Rat Na V 1.4 channels and mutants thereof were expressed in mammalian cells and were assayed with the whole-cell patch-clamp method. Unlike for the M-superfamily µ-conotoxin GIIIA from Conus geographus, channel block by MrVIA was strongly diminished after activating the Na V channels by depolarizing voltage steps. Searching for the source of this voltage dependence, the gating charges in all four-voltage sensors were reduced by site-directed mutagenesis showing that alterations of the voltage sensor in domain-2 have the strongest impact on MrVIA action. These results, together with previous findings that the effect of MrVIA depends on the structure of the pore-loop in domain-3, suggest a functional similarity with scorpion b-toxins. In fact, MrVIA functionally competed with the scorpion b-toxin Ts1 from Tityus serrulatus, while it did not show competition with µ-GIIIA. Ts1 and µ-GIIIA did not compete either. Thus, similar to scorpion b-toxins, µO-conotoxins are voltage-sensor toxins targeting receptor site-4 on Na V channels. They "block" Na + flow most likely by hindering the voltage sensor in domain-2 from activating and, hence, the channel from opening.
Biophysical Journal, 1994
The rat brain la (Brila) Na channel a-subunit and the brain 1 subunit were coexpressed in Xenopus oocytes, and peak whole-oocyte Na current (I) was measured at a test potential of-10 mV. Hyperpolarization of the holding potential resulted in an increased affinity of STX and TTX rested-state block of Brila Na channels. The apparent half-block concentration (EDJ) for STX of Brila current decreased with hyperpolarizing holding potentials (Vhm). At Vhw of-100 mV, the ED50 was 2.1 + 0.4 nM, and the affinity increased to a ED5,of 1.2 0.2 nM with Vhm of-140 mV. In the absence of toxin, the peakcurrent amplitude was the same for all potentals negative to-90 mV, deonsstrating that all of the channels were in a closed conformation and maximally available to open in this range of holding potentals. The Woodhull model (1973) was used to describe the increase of the STX ED5 as a funcbon of holding potential. The equivalent elctrcl distance of blck (8) by STX was 0.18 from the exracellular milieu when the valence of STX was fixed to +2. Analysis of the hoding potential dependence of TTX blck yiekled a similar 8 when the valence of TTX was fixed to + 1. We conclude that the guanidinium toxin site is located partially within the bansmembrane elkctric field. Previous site-directed mutagenesis shtdies demonstrated that an isoforn-specific phenylalanine in the BrIla channel is crical for high affinity toxin block. Therefore, we propose that amino acids at positions corresponding to this Phe in the Brila channel, which lie in the outer vestibule of the channel adjacent to the pore entance, are partially in the transmembrane potential drop.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2009
Gangliosides are glycolipids mainly present at the plasma membrane (PM). Antibodies to gangliosides have been associated with a wide range of neuropathy syndromes. Particularly, antibodies to GM1 ganglioside are present in patients with Guillain-Barré syndrome (GBS). We investigated the binding and intracellular fate of antibody to GM1 obtained from rabbits with experimental GBS in comparison with the transport of cholera toxin (CTx), which binds with high affinity to GM1. We demonstrated that antibody to GM1 is rapidly and specifically endocytosed in CHO-K1 cells. After internalization, the antibody transited sorting endosomes to accumulate at the recycling endosome. Endocytosed antibody to GM1 is recycled back to the PM and released into the culture medium. In CHO-K1 cells, antibody to GM1 colocalized with coendocytosed CTx at early and recycling endosomes, but not in Golgi complex and endoplasmic reticulum, where CTx was also located. Antibody to GM1, in contraposition to CTx, showed a reduced internalization to recycling endosomes in COS-7 cells and neural cell lines SH-SY5Y and Neuro2A. Results from photobleaching studies revealed differences in the lateral mobility of antibody to GM1 in the PM of analyzed cell lines, suggesting a relationship between the efficiency of endocytosis and lateral mobility of GM1 at the PM. Taken together, results indicate that two different ligands of GM1 ganglioside (antibody and CTx) are differentially endocytosed and trafficked, providing the basis to gain further insight into the mechanisms that operate in the intracellular trafficking of glycosphingolipid-binding toxins and pathological effects of neuropathyassociated antibodies.
Brain Research, 1986
Different domains of the sodium channel were characterized in rat sciatic nerve according to the binding of antibodies to their antigenic determinants. An extracellular domain, accessible to antibodies, modulates channel conductance. Another domain, which is not involved in the physiological activity, becomes accessible to externally applied antibodies only after prolonged exposure. This study also revealed mobile antigenic determinants whose internalization can be detected by immunofluorescence. A number of sodium channel-specific monoctonal antibodies (mAb's) have previously been characterized 1-4,7-11,14. Many of these were generated against eel sodium channell,3, 4,7,9,l°. In preparation of the latter, two immunogens were used: the solubilized and partially purified tetrodotoxin (TTX)-receptor 3,4,10,11 and sodium channel rich membrane fragments of eel electroplaxS,10,11. The mAb's 5D~0 and 5F 3 belong to the first group. They were selected for their ability to bind 3,4 and immunoprecipitate 1°.11 the TTX-receptor. These mAb's appear to bind specifically to the sodium channel in fish preparations 4,5,
Comparative distribution of voltage-gated sodium channel proteins in human brain
Molecular Brain Research, 2001
Antisera directed against unique peptide regions from each of the human brain voltage-gated sodium channel a subunits were generated. In immunoblots these were found to be highly specific for the corresponding recombinant polypeptides and to recognise the native holoprotein in human brain membrane preparations. These antisera were used to perform a comparative immunohistochemical distribution analysis of all four brain sodium channel subtypes in selected human CNS regions. Distinct but heterogeneous distribution patterns were observed for each of the a subunits. In general, these were complimentary to that previously shown for the corresponding human mRNAs. A high degree of conservation with respect to the distribution found in rat was also evident. The human a subunit proteins exhibited distinct subcellular localisation patterns. Types I, III and VI immunoreactivity was predominantly in neuronal cell bodies and proximal processes, whereas type II was concentrated along axons. This is similar to rat brain and suggests the different the sodium channel subtypes have distinct functions which are highly conserved between human and rodents. A notable difference was that the type III protein was detected in all human brain regions examined, unlike in rat brain where expression in adults is very restricted. Also in contrast to rat brain, the human type VI protein was not detected in axons of unmyelinated neurons. These differences may reflect true species variation and could have important implications for understanding the function of the sodium channel subtypes and their roles in human disease.