Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using 125I-alpha scorpion toxin. II. Sodium distribution on postsynaptic membranes

Abstract

A radioiodinated alpha-scorpion toxin (toxin II from Androctonus australis Hector) (alpha ScTx) was used as a probe for EM autoradiography to study the distribution of voltage-dependent sodium channels (Na+ channel) on the postsynaptic side of the mouse neuromuscular junction. Silver grain distribution was analyzed by the cross-fire method to assess the relative Na+ channel density in each membrane domain measured by stereology. This analysis showed that the maximum Na+ channel density was located on the edge of the synaptic gutter, where it reached about twice the mean density in the postsynaptic fold membrane. Na+ channel densities have been calculated using ACh receptor (AChR) density in fold crests as reference. Sodium channel density on the edge of the synaptic gutter was estimated at about 5000/microns 2. Sodium channel distribution in the postsynaptic folds was compared to AChR distribution using density distribution analysis (Fertuck and Salpeter, 1976). The results confirmed that, as already observed by immunogold labeling (Flucher and Daniels, 1989), there are no Na+ channels on fold crests. Na+ channels are located in the rest of the fold membrane (bottom) and may be distributed according to two possible models. In the first, density would be uniformly high, although lower than on the gutter edge. In the second, density would decrease from the crest border, where the value was that of the gutter edge, to the fold end, where the value would be 50% lower. Based on the latter model, which was the “best-fit model,” we propose that the postsynaptic membrane includes two domains. The first is the fold crest, which contains almost exclusively AChRs. This domain is devoted to reception-transduction of the chemical signal. The second includes both the fold bottom membrane and the perisynaptic membrane. Sodium channel density is highest along the crest border and decreases moving away. Its functions are the integration of postsynaptic potentials and generation-conduction of the muscle action potential.

Action potential generation in rat slow‐ and fast‐twitch muscles.

“…In the present study, the threshold depolarization evoked by current injection was consistently several millivolts lower at the NMJ than in the extrajunctional region in both the soleus and the EDL (at least at a resting potential of -90 mV, see below). Considering the substantial increase in sodium channel density (5-to 10-fold: Beam et al 1985;Caldwell et al 1986;Flucher & Daniels, 1989;Boudier et al 1992) at the NMJ and the extensive membrane folding that characterizes vertebrate endplates (Salpeter, 1987), it seemed surprising that the threshold should be lowered by only a few millivolts. A much lower value for threshold at the NMJ was observed when values obtained from nerve-evoked action potentials were considered (Fig.…”

“…Estimates of sodium channel density by Boudier et al (1992) indicated that there was a tenfold increase per unit area of membrane at the NMJ compared with elsewhere on the muscle fibre. Accordingly, the high density of sodium channels at the NMJ arises from an increased number of channels per unit area of membrane at the junction and an increase in the amount of membrane as a result of the highly folded postsynaptic structure.…”

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

“…He found a twofold difference in capacitance between end plate and extrajunctional membrane; this would mean that postjunctional folding could account for at most a twofold increase in current density at the synapse, assuming that NaChs were distributed uniformly in the postjunctional membrane. (2) Flucher and Daniels (1989; see also Boudier et al, 1992) used immunocytochemistry to show that NaChs were not distributed uniformly in the postjunctional membrane, but were concentrated in the troughs and nearly absent from the crests of the folds, which has been estimated to comprise -30% of the postsynaptic surface area (Matthews-Bellinger and Salpeter, 1983). Thus, the effect of membrane folding on current density measurements in end plate patches is reduced to approximately 1.7-fold.…”

“…If channels were uniformly distributed, this twofold increase in membrane area would double the apparent NaCh density. Second, electron microscopic studies using immunocytochemistry (Flucher and Daniels, 1989) and radiolabeled scorpion toxin (Boudier et al, 1992) indicate that NaChs are concentrated in the depths of the postsynaptic folds and are absent or at low density on the crests of the folds where AChRs are concentrated. Thus, NaChs are segregated within postsynaptic membrane.…”

Kir4.1 and AQP4 associate with Dp71‐ and utrophin‐DAPs complexes in specific and defined microdomains of Müller retinal glial cell membrane

“…Sodium channels are also concentrated at the NMJ where they are restricted to the depth of the postjunctional folds (Boudier et al, 1992). At the NMJ reside the dystrophin/utrophin and their associated protein complexes (DAPs) that serve as molecular link between the cytoskeleton and the surrounding extracellular matrix, providing mechanical stability to the NMJ components (Deconinck et al, 1997).…”