Measurements of intracellular ionized calcium in squid giant axons using calcium-selective electrodes (original) (raw)

Ionized calcium concentrations in squid axons

Journal of General Physiology, 1976

AaSTRACT Values for ionized [Ca] in squid axons were obtained by measuring the light emission front a 0.1-9.1 drop of aequorin confined to a plastic dialysis tube of 140-/zm diameter located axially. Ionized Ca had a mean value of 20 × 10 -y M as judged by the subsequent introduction of CaEGTA/EGTA buffer (ratio ca. 0.1) into the axoplasm, and light measurement on a second aequorin drop. Ionized Ca in axoplasm was also measured by introducing arsenazo dye into an axon by injection and measuring the Ca complex of such a dye by multichannel spectrophotometry. Values so obtained were ca. 50 × 10 -9 M as calibrated against CaEGTA/ EGTA buffer mixtures. With a freshly isolated axon in 10 mM Ca seawater, the aequorin glow invariably increased with time; a seawater [Ca] of 2-3 mM allowed a steady state with respect to [Cain. Replacement of Na + in seawater with choline led to a large increase in light emission from aequorin. Li seawater partially reversed this change and the reintroduction of Na + brought light levels back to their initial value. Stimulation at 60/s for 2-5 rain produced an increase in aequorin glow about 0.1% of that represented by the known Ca influx, suggesting operationally the presence of substantial Ca buffering. Treatment of an axon with CN produced a very large increase in aequorin glow and in Ca arsenazo formation only if the external seawater contained Ca.

The influence of chemical agents on the level of ionized [Ca2+] in squid axons

The Journal of General Physiology, 1985

Squid giant axons injected with either aequorin or arsenazo III and bathed in 3 mM Ca (Na) seawater were transferred to 3 mM Ca (K) seawater and the response of the aequorin light or the change in the absorbance of arsenazo III was followed. These experimental conditions were chosen because they measure the change in the rate of Na/Ca exchange in introducing Ca into the axon upon depolarization ; [Cal. is too low to effect a channel-based system of Ca entry. This procedure was applied to axons treated with a variety of compounds that have been implicated as inhibitors of Na/Ca exchange. The result obtained was that the substances tested could be placed in three groups. (a) Substances that were without effect on Ca entry effected by Na/Ca exchange were : D600 at 10-100 FM, nitrendipine at 1-5 uM, Ba2+ and Mg" at concentrations of 10-50 mM, lidocaine at 0.1-10 mM, cyanide at 2 mM, adriamycin at a concentration of 3,M, chloradenosine at 35 ,AM, 2,4-diaminopyridine at 1 mM, Cs' at 45-90 mM, and tetrodotoxin at 10'. (b) Substances that had a significant inhibitory effect on Na/Ca exchange were : Mn2+ , Cd2+ , and Las" at 1-50 mM, and quinidine at 50 pM. (c) There were also blocking agents and biochemical inhibitors whose action appeared to be the inhibition of nonmitochondrial Ca buffering in axoplasm rather than an inhibition of Na/Ca exchange. These were the general anesthetic 1-octanol at 0.1 mM and 1 mM orthovanadate plus apyrase.

Calcium efflux from internally dialyzed squid axons: The influence of external and internal cations

Journal of Supramolecular Structure, 1974

Internal dialysis techniques have been used t o examine the influence of external and internal cations o n Ca efflux from ATP-depleted squid axons. The main observation is that Ca efflux is promoted by external Na and inhibited by internal Na. The Nao-dependent Ca efflux appears t o be a function of [ Nal i , and is also affected by the membrane potential; a 25 mV depolarization may cause as much as an e-fold decrease in Ca efflux. These data are consistent with a counter-transport exchange of 3Na+-for-1Ca2+. A Cao-dependent Ca efflux has also been observed; it is prominent in Na sea water or Li sea water, and is markedly diminished in choline sea water. This flux is consistent with the idea of a Ca-Ca exchange diffusion process. Taken together, the Nao-and the Cao-dependent Ca effluxes fit a two-site model for carrier-mediated Ca transport; one site binds two Na+ o r one Ca2+, while the second site can bind either one Na+ or one Li' . The data reported here suggest that both sites must be filled on the inward journey, but that only the Ca-binding site need be occupied on the outward journey of the carrier. A mechanism of this type could derive sufficient energy from the Na and voltage gradients t o maintain a [Ca2+]o/[Ca2+]i concentration ratio of about lo4 in the absence of ATP. The present experiments d o not, however, rule out the possible participation of a metabolically driven Ca transport mechanism in vivo.

The control of ionized calcium in squid axons

Journal of General Physiology, 1977

A n S TR A C T Measurements of the Ca content, [Ca}r, of freshly isolated squid axons show a value of 60 p, mol/kg axoplasm. Axons in 3 mM Ca (Na) seawater show little change in Ca content over 4 h, while axons in 10 mM Ca (Na) seawater show gains of 18/zmol Ca/kg-h. In 10 Ca (choline) seawater the gain is 2,400/~mol/kg. h. Using aequorin confined to a dialysis capillary in the center of an axon, one finds that [Ca~ is in a steady state with 3 Ca (Na) seawater, and that both 10 Ca (Na) and 3 Ca seawater cause increases in [Ca]l. In 3 Ca (Na) seawater-3 Ca (choline) seawater mixtures, 180 mM [Na]0 (40% Na) is as effective as 450 mM [Na]0 (100% Na) in maintaining a normal [Ca]; lower [Na] causes an increase in [Cal. If axons are injected with the ATP-splitting enzyme apyrase, the resulting [Call is not different from control axons. Such axons also recover to their initial [Ca~ after loading with high [Ca]o or low [Na]o solutions. Depolarization of an axon with 100 mM K (Na) seawater leads to an increase in the steady-state level of [Ca]~ that is reversed upon returning the axon to normal seawater. Freshly isolated axons treated with either CN or FCCP to inhibit mitochondrial Ca buffering can still maintain a normal [Ca~ in 1 Ca (Na) seawater.

Calcium measurement in the periphery of an axon

The Journal of General Physiology, 1979

A B S T g A C T Aequorin was microinjected into squid giant axons, the axons were stimulated, and the change in light emission was followed. This response was compared with that found when the axon, in addition to being microinjected with aequorin, is also injected with the dye phenol red. Large concentrations of phenol red injected into axons result in a high probability that photons emitted by aequorin, when it reacts with Ca in the core of the axoplasm, will be absorbed before they escape from the axon; photons produced by the aequorin reaction at the periphery of the axoplasm are much less likely to be absorbed. This technique thus favors observing changes in Ca, taking place in the periphery of the axon. Stimulation in 50 mM Ca seawater of an aequorin-phenol red-injected axon at 180 s-a for 1 min produces a scarcely detectable change in Ca,; the addition of 2 mM cyanide (CN) to the seawater produces an easily measureable increase in Cai, suggesting that mitochondrial buffering in the periphery is substantial. Making the pH of the axoplasm of a normal axon alkaline with 30 mM NH~'-50 mM Ca seawater, reduces the resting glow of the axon but results in an even more rapid increase in Ca, with stimulation. In a phenol red-injected axon, this treatment results in a measureable response to stimulation in the absence of CN.

Dependence of Ionized and Total Ca in Squid Axons on Naofree or High=Ko Conditions

A BS T R A C T The level of intracellular Ca in squid axons (both ionized and total Ca) was studied as a function of the experimental variables [Na];, [Na]o, pHi , cyanide, and depolarization . Ionized Ca was measured by following the light emission of aequorin while total Ca was measured by the atomic absorption analysis of samples of axoplasm . Aequorin glow is known to be increased either by the application of Na ofree solutions or by depolarization produced by external solutions containing greater than normal K concentrations . The present results show that if [Na] i is low, the depolarization that is brought about by solutions with elevated [K] leads to a resting light emission that is decreased rather than increased, as is the case when [Na]i is high . In axons where [Na] i is varied, a comparison of the increments in light emission produced by the application first of Na-free and then of high-K solutions shows that they have an identical dependence on [Na] ;, with a half-activation of Ca entry produced by an [Na] i of 25-30 mM . Changes in pH ; affect the aequorin signal produced by depolarization, with acidification reducing and alkanization increasing the response. Cyanide did not greatly affect the size of the signal resulting from either Na removal or that from depolarization .

__ Big Calcium Summary Journal of General Physiology JGP with Cover May 2009 comp3.pdf

Voltage-dependent calcium channels relay electrical signals from the cell membrane to the cytoplasm by allowing extracellular Ca 2+ to flow into the cell, where Ca 2+ acts as a chemical signal . The Ca 2+ -conducting pore of these transmembrane proteins selects Ca 2+ over the much more abundant cations Na + and K + . Four amino acid residues bearing acidic side chains (the "EEEE" locus in L-type calcium channels) extend into this pore, and all cooperate in selecting Ca 2+ with high affinity for review see . Here, we compute consequences of a physical model of selectivity in which ions compete for interaction with negative structural charge and for restricted space in the EEEE locus.

Mechanisms of calcium transport in the giant axon of the squid and their physiological role

Cell Calcium, 1980

The giant axon of the squid has been extensively used as a model for studying Ca regulation in excitable cells. Different techniques (extrusion, injection and dialysis) have been employed to characterize Ca fluxes across the axon membrane. [Ca2+lip Since both Ca efflux and influx are markedly dependent on considerable effort has been dedicated to determine the resting value of the [Ca'+]-Results from different laboratories indicate that the [Ca'+]i, in a normal f&e, range from 20-100 I-&. control), with an imposed [Ca2+]i Under dialysis conditions (internal of 80 nM, Ca influx is balanced by an outward Ca movement of about 40 f/CS. Ca extrusion occurs through two parallel transport systems: one having a high affinity for [Ca2+]i, dependent on ATP, not affected by Nai, Na,, Ca,, Mgo and inhibited by internal vanadate (uncoupled component), the other, more prominent at relatively high [Ca2+]i,does not require ATP, is inhibited by Nai activated by Na, and not inhibited by vanadate. (Nao-dependent component). The existence of these two systems provide the axon with an effective way to maintain in the long term a constant low [Ca2+]i in spite of short term fluctuations due to increased Ca influx during nervous activity.