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

Abstract

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.

Voltage-Gated Sodium Channels and AnkyrinG Occupy a Different Postsynaptic Domain from Acetylcholine Receptors from an Early Stage of Neuromuscular Junction Maturation in Rats

“…However, these antibodies did not distinguish between the known isoforms of these proteins. Previous studies, using isoformspecific antibodies for Na V 1 and syntrophins, suggest that in newborn muscles the isoforms characteristic of the adult NMJ (Na V 1.4, ␤ 2 -syntrophin) are present in very low amounts, if at all (Lupa et al, 1993;Kramarcy and Sealock, 2000). Therefore, we believe that the major isoforms of these proteins that we have detected in newborn muscles are Na V 1.5 and ␣-syntrophin.…”

“…Because an antibody that labels Na V 1.5 specifically is not available, we also used an antibody specific for Na V 1.4. No labeling was seen in sections of muscles at P0 (data not shown), but it was present from P14 on, confirming the low levels of Na V 1.4 early in development (Lupa et al, 1993). Thus, at birth, most of the Na V 1 we detected in the perijunctional region using an antibody that recognizes all forms of Na V 1 (DugandzijaNovakovic et al, 1995) was Na V 1.5.…”

“…We have shown that the well defined clusters of Na V 1 and ankyrinG characteristic of the mature NMJ first appear when the main phase of postsynaptic fold formation nears completion (see also Lupa et al, 1993). A combination of localized synthesis and exclusion of Na V 1 from AChR-rich regions could, in principle, account for the high density of Na V 1 in the depths of the folds at mature NMJs.…”

“…Figure 2 A shows that 1-2 weeks after birth Na V 1 labeling was apparent at the NMJ and by P28 resembled the adult distribution. Previous studies have shown that Na V 1.4, the adult form of muscle Na V 1, does not become concentrated in the postsynaptic membrane of rat NMJs until 2-3 weeks after birth (Lupa et al, 1993). We were interested to know whether there was an earlier accumulation of Na V 1.5, the fetal form.…”

“…In contrast, Na V 1 channels cannot be detected using immunolabeling techniques until birth, when the pattern of labeling is suggestive of a diffuse zone of increased density in the vicinity of the NMJ . During the next 1-2 weeks, when the postsynaptic folds are forming (Bewick et al, 1996), the highly localized accumulation of Na V 1 channels characteristic of the mature NMJ develops (Lupa et al, 1993), and Na V 1 channels also appear along the rest of the muscle fiber surface Stocksley and Slater, 1999). These observations have led to the suggestion that Na V 1 channel clustering, unlike AChR clustering, is dependent on postsynaptic fold formation.…”