Synthesis of sodium channels in the cell bodies of squid giant axons.

Abstract

Giant axons in squid are formed by fusion of axons from many small cell bodies in the giant fiber lobe (GFL) of the stellate ganglion. Somata of GFL cells in vivo are inexcitable and do not have measurable sodium current (INa) when studied with microelectrode or patch-electrode voltageclamp techniques. If GFL cells are separated from the giant axons and maintained in primary culture, axon-like INa can be recorded from the somata after several days. Incorporation of Na channels into GFL cell bodies requires protein synthesis, intracellular microtubule-based transport, and the lack of a morphologically defined axon to serve as a sink for channels synthesized in culture.Voltage-controlled Na channels are integral membrane glycoproteins that mediate the electrogenic flow of extracellular Na ions into animal cells and subserve the propagation of action potentials used by neurons for rapid signalling via axons (1). Functional aspects of these proteins (2, 3) and primary structure (4, 5) are known in detail; biochemical (1, 6) and higher-order structural (7) characterization are rapidly progressing. Considerably less is known about the biosynthesis, distribution, and degradation of Na channels, and studies of these phenomena have utilized biochemical (8, 9) and morphological (10) approaches.Voltage-clamp methods provide another excellent probe for detecting functional Na channels because of the high degree of resolution provided (11, 12). Such measurements have been used to map Na channel distribution in skeletal muscle fibers (13,14) and to assess the mobility of channels in the plasma membrane (15). We have undertaken an electrophysiological approach to begin studying the dynamic features of Na-channel cell biology in the numerous cell bodies in the giant fiber lobe (GFL) of the squid's stellate ganglion, which give rise to the giant axon through fusion of their axonal initial segments (16). This system is morphologically simple, experimentally accessible, and the axon has been the subject of extensive investigation (17).Although the many small GFL somata must normally synthesize Na-channel proteins for the giant axons (18), they are inexcitable themselves (19). We find that if these cell bodies are dissociated from the giant axons and maintained in primary culture, Na current (INa) appears within several days. This condition persists for at least 2 weeks and is inhibited by actinomycin D or by demecolcine, a colchicine derivative. Our results suggest that an important step leading to the appearance of operational Na channels in the soma membrane involves intracellular translocation of newly synthesized channels along microtubule pathways. MATERIALS AND METHODSCell bodies (diameter, 20-40 ,um) were isolated from the posterior tip of the giant fiber lobe of stellate ganglia (16,20) in Loligo opalescens and Lolliguncula brevis (21) and maintained in primary culture at 15'C essentially as described by Bookman and coworkers (22,23). Excised GFL tips were treated with nonspecific protease (10 mg/ml, type XIV; Sig...

Localization of voltage-gated K+ channels in squid giant axons

“…In particular, fast activating sodium ion channels underlie the rapid upstroke phase of the action potential and potassium ion channels underlie its repolarization phase. A similar conclusion applies for sodium ion channels (Brismar and Gilly, 1987). In particular, mRNA for the potassium ion channel has been found with in situ hybridization to occur solely in the cell body and not in the axon itself or in the glial cells which surround the axon (Rosenthal et al, 1996).…”

“…As with most proteins, these channels appear to undergo recycling, or turnover, in this case between the axolemma and the neuronal cell body. Both ion channel types are packaged in small vesicles in the soma, which then traffic down the axon (Brismar and Gilly, 1987;Wonderlin and French, 1991). A similar conclusion applies for sodium ion channels (Brismar and Gilly, 1987).…”

High-fidelity patch-clamp recordings from neurons cultured on a polymer microchip

“…This is consistent with reports of low ion channel density on neuronal cell bodies isolated from the snail Helix pomatia, where calcium channel density was 1 per 3 µm 2 (Lux et al 1984), or in the soma and the growth cones of Lymnaea neurons where the density of stretch-activated channels selective for K + was 1 per 1-2 µm 2 (Sigurdson and Morris 1989). These values are very low when compared to the Na + channel density in the squid axon (166-533 per µm 2 ; Hille (1984)) or in squid stellate ganglion cell bodies (37 per µm 2 ; Brismar and Gilly (1987)). From these background recordings, we calculated the standard deviation of the baseline current to be 0.7 pA: the minimum signal distinguishable from the baseline fluctuation at 95% confidence level is then 2.1 pA, within the resolution of single ion channel current jumps.…”

Elevated ankyrin G in a plexiform neurofibroma and neuromas associated with pain

“…At this point, evidence indicates the involvement of peripheral nerve injury, axonal sprouting, and failed nerve regeneration. 3,8,10,17 Neoplastic transformation of a peripheral nerve could, however, by yet undetected mediators incite such an axolemmal change as well. Recent highly specific voltage gated Na + channel insertion into the axolemma (e.g.…”