Association of Calcium With Membranes of Squid Giant Axon

Abstract

Giant axons from the squid, Loligo pealei, were fixed in glutaraldehyde and posttixed in osmium tetroxide. Calcium chloride (5 mM/liter) was added to all aqueous solutions used for tissue processing. Electron-opaque deposits were found along the axonal plasma membranes, within mitochondria, and along the basal plasma membranes of Schwann ceils. X-ray microprobe analysis (EMMA-4) yielded signals for calcium and phosphorus when deposits were probed, whereas these elements were not detected in the axoplasm. I N T R O D U C T I O NMany cellular processes appear to be mediated by reversible interactions of ionized calcium with the plasma membrane. We have been interested in using the electron microscope to locate membrane areas with high affinity for calcium. Addition of calcium ions to the solutions used for fixing and processing tissues for the electron microscope causes opaque deposits to be formed along the plasma membranes of a variety of ceils (I). In insect intestine, for example, deposits occur on apical membranes (microvilli) and along septate but not along gap junctions (2). Apparently, particular regions of the plasma membrane, or some structure closely associated with the membrane, can sequester enough calcium to make the region adjacent to the membrane opaque to electrons. Although we do not know precisely how the deposits form, we suspect that the presence of calcium ions during processing keeps intraceUular binding sites saturated. The deposits were not observed readily in the past because of the widespread use of osmium, which decalcifies tissues unless excess calcium is present in the fixative.To assess the significance of the deposits adjacent to the membranes, one must know their composition and how they form. The present study was undertaken to utilize the rapidly developing technique of electron-probe X-ray microanalysis (3, 4) to study the composition of the deposits. The analytical electron microscope is a transmission electron microscope fitted with X-ray spectrometers that analyze X rays generated when a micro-area of the specimen is bombarded by the electron beam. Since each element produces X rays of characteristic energy and wavelength, information is obtained about the composition of the specimen. Microprobe analysis is well suited for analyzing bound ions, since conventional fixation and embedding techniques can be used.

Structural complexes in the squid giant axon membrane sensitive to ionic concentrations and cardiac glycosides

“…Note that the structural complexes described in the present work are different from the electrondense deposits found by Oschman et al (10) in the giant axon of squid Loligo pealii and by Hillman and Llinhs (9) in the giant synapse of the same squid. These differences are: (a) the electron-dense deposits were found when the nerves were fixed in glutaraldehyde containing 5 mM CaC12, and were not found when the tissue was prefixed in OsO, (9), whereas the structural complexes described in the present work are observed in nerve fibers fixed in OsO,, and, furthermore, they remain relatively unmodified by variations in the external calcium concentration.…”

“…Besides, these dense deposits appear as globules or plaques formed by subunits; and the three-laminar substructure of the axolemma and the narrowing of the intercellular space have not been pointed out in connection with them. Note, however, that electron-dense deposits similar to those of Oschman et al (10) and Hillman and Llin/ls (9) appeared erratically in certain of our nerve halves (see Results and Fig. 6).…”

“…In some of the nerve halves incubated in low sodium and low potassium sea water, as well as in some of their control halves, electron-dense deposits, similar to those induced by calcium as reported by Oschman et al (10), were occasionally observed (Fig. 6).…”

“…Other types of dense structures, such as the deposits shown by Oschman et al (10) and Hillman and Llin~s (9) in the presence of high calcium, were not observed in the groups of nerve fibers incubated with or without calcium. They were not observed in the nerves incubated in the presence of magnesium either, nor even in those two groups of nerves which, in a confirming experiment, were incubated in the presence of 44 mM Ca added to a normal magnesium concentration (53 mM).…”

On the role of mitochondria in transmitter release from motor nerve terminals.

“…(The qualitative interpretation of the experimental results will be similar if we assume that release of transmitter depends on the surface density of calcium ions on the inside of the presynaptic membrane; which in turn will depend on the free intracellular calcium concentration.) The free intracellular calcium concentration will depend on a balance between several processes: (1) the resting calcium influx and the calcium influx following depolarization (Baker, Hodgkin & Ridgway, 1971) which raises [Ca]in; (2) the opposing calcium efflux (Baker & Blaustein, 1968;Blaustein & Hodgkin, 1969;Baker, Blaustein, Hodgkin & Steinhardt, 1969;DiPolo, 1973;Mullins & Brinley, 1975) which lowers [Ca]in; (3) intracellular calcium buffers such as the mitochondria (Lehninger, 1970); and other calcium binding systems (see Hillman & Llinas, 1974;Oschman, Hall, Peters & Wall, 1974), that are able to bind calcium reversibly. Thus, the regulation of the intracellular calcium 298 MITOCHONDRIA AND TRANSMITTER RELEASE 299 concentration is a complex process, depending on the contributions of the various components (see schematic representation in Fig.…”