Faculty Bibliography

Hydration of single or mixed phospholipids or lipid protein mixtures at low ionic strength results in the formation of a population of large, solvent free, single bilayer vesicles with included volumes of up to 300 microliters/mumol lipid. Their size ranges from 0.1 to 300 microns and they can be sorted out according to size by centrifugation. When formed in distilled water their internal solution has a conductivity of 20-50 microseconds/cm-1, an osmolarity of 0.5-5 mOsM, and a density of 1.0005-1.001. The osmotic pressure produced by the internal solutes cause a surface stress of 25 dyn/cm for a 20-microns vesicle. Their elastic constant ranges from 75-150 dyn/cm. During formation they can internalize particles such as latex beads or cell nuclei. They can be impaled with microelectrodes, or patch clamped. They can also be sealed to a small Vaseline-treated hole in a thin partition between two aqueous compartments. Sealing occurs in two stages. In the first stage sealing resistance is similar to that seen with patch-clamp pipettes. In the second stage, a much tighter seal is obtained. After sealing, the smaller portion of the sealed vesicle can be selectively broken by an electric shock leaving a single membrane across the hole. The capacitance and resistance of such membranes, in the presence of 10 mM NaCl, are approximately 0.7 microF/cm2 and 10(8) omega cm2 for pure lipid vesicles. Gramicidin increases the membrane conductance and monazomycin induces voltage-dependent gating thus providing further evidence that the vesicles are bounded by a single bilayer

In this paper, we describe a simple and highly sensitive manual assay for isotope fluxes through ion-conducting pathways, particularly cation-specific channels, in heterogenous populations of small membrane vesicles. We measure uptake of tracer of the ion of interest, against a large chemical gradient of the same ion. As a result of the imposed chemical gradient, a transient electrical diffusion potential is set up across the membranes of those vesicles which are highly permeable to the ion of interest. The isotope tends to equilibrate with the diffusion potential and is therefore concentrated selectively and transiently into those vesicle containing the channels. Furthermore, when performed in this way, the time course of tracer equilibration occurs over several minutes, rather than the sub-second range expected for tracer equilibration into channel-containing vesicles in the absence of an opposing chemical gradient of the permeant ion. The use of the procedure is demonstrated for three Na-conducting channels: gramicidin D incorporated into phospholipid vesicles, amiloride-blockable Na channels in toad bladder microsomes, and veratridine-activated tetrodotoxin-blockable Na channels in rat brain synaptic membranes. For all three cases, it proved simple to measure a specific 22Na uptake, in a minute time range, using very low concentrations of the channel-containing vesicles. By comparison with isotope flux measurements performed without an opposing Na gradient, the power of the present assay derives from both the very large gain in sensitivity and the convenient time course

The PC12 clone is a line of rat pheochromocytoma cells which undergoes neuronal differentiation in the presence of nerve growth factor (NGF) protein. In the absence of NGF, PC12 cells are electrically inexcitable, while after several weeks of NGF treatment, they develop sodium action potentials. The number and density of sodium channels on PC12 cells before and after treatment with NGF were estimated by measuring the binding of [3H]saxitoxin ([3H]STX). The data indicate that [3H]STX binding increases in the NGF-treated cells by 15- to 20-fold per cell, 3- to 10-fold per mg of protein, and an estimated 7-fold per unit area of membrane. The kinetic properties for [3H]STX binding are unchanged, however, by NGF treatment. A Hodgkin-Huxley analysis (Hodgkin, A. L., and A. F. Huxley (1952) J. Physiol. (Lond.) 117: 500-544) suggests that the estimated density of sodium channels in NGF-untreated PC12 cells is sufficient to explain their lack of excitability. On the other hand, the estimated channel density on the NGF-treated cells (30 to 50/micrometers 2) is comparable to that in other excitable systems. Thus, the development of excitability in PC12 cells in response to NGF could be due to the induction of sodium channel synthesis

1. The action potential in Myxicola giant axons is abolished if the nerve is stimulated at frequencies higher than about 5 sec-1. At 1 sec-1 the magnitude of the action potential is not maintained upon sequential stimulation but decreases until the response is abolished. 2. The behaviour of the ionic currents underlying the action potential was studied with voltage-clamp techniques to find the origin of such adaptation. These studies showed a frequency-dependent decline of the sodium currents. 3. The decline in the Na currents upon repetitive depolarization is shown to be due to a decrease in the Na conductance and not to change in driving force. 4. An analysis of the effects of conditioning depolarizations on the Na current during a depolarizing test pulse demonstrates that in a single short depolarization (less than 10 msec) 15% of the Na conductance enters an inactivated state from which recovery is very slow. Upon repetitive depolarizations the amount of Na conductance available accumulates in this slowly recovering inactivated state. 5. The data are explained by proposing that every time the membrane is depolarized open channels undergo one of two competing reactions. Open channels enter either the traditional inactivated state described by Hodgkin & Huxley (1952b) from which recovery is fast (a few milliseconds) or an inactivated state from which recovery is very slow (seconds). In Myxicola, only 15% of open channels enter the later inactivated state in a single depolarization. Upon repetitive depolarizations, however, the fraction in this state accumulates if the frequency of pulsing is faster than the rate of recovery. 6. Axons in which the amount of open channels entering the slowly recovering inactivated state is significant, such as in Myxicola, have thus a system capable of storing the previous activity of the axon for periods of seconds or minutes

1. Squid giant axons internally perfused with CsF have their Na conductance inactivated due to the low value of the resting potential. When hyperpolarized with voltage clamp to normal values of resting potential, the Na conductance recovers with an exponential time course. The time constant of recovery is of the order of 30 sec at a membrane potential of -70 mV and at 5 degrees C. The recovery from slow inactivation has a Q10 of about 3. 2. The development of inactivation during depolarization is also slow. The time constant varies between 10 and 20 sec at 5 degrees C, depending upon the value of the membrane potential. 3. Slow inactivation is also observed in NaF perfused axons and in intact axons with a low resting potential. 4. Although internal perfusion with pronase (or a purified fraction of this enzymic complex) blocks the fast (h) inactivation of the Na conductance, the slow inactivation remains. The recovery is similar before and after the proteolytic treatment. However, slow inactivation appears to develop faster after enzymic perfusion. 5. Slow inactivation develops without any apparent change in distributed or local membrane surface charge. 6. The experiments suggest that slow inactivation is a general property of the Na conductance as in many other conductance channels in excitable membranes. The experiments can be interpreted by proposing that slow inactivation is a phenomenon independent of fast inactivation, and that pronase somehow accelerates the onset of slow inactivation. 7. An alternative model, in which slow inactivation is coupled to fast inactivation, is proposed. This model is consistent with the results presented here and is very similar to one proposed to explain the frequency response of the sodium currents in Myxicola giant axons (Rudy, 1975, 1978)

Intracellular perfusion of giant axons from Loligo forbesi with a crude protein extract of Pronase dissolved in a KF solution suppresses the process of fast inactivation of the Na conductance (the h-process in the Hodgkin-Huxley terminology). 2. The results with protease inhibitors indicate that the most substrate specific endopeptidase present in pronase, alkaline proteinase b, destroys the h-process. 3. After destruction of the inactivation the conductance rise upon depolarization followed cube law kinetics. Values of the time constant taum before and after destruction of the h-process were very similar. 4. After destruction of the inactivation process the following properties were tested: cation selectivity, instantaneous conductance and internal receptor sites for tetrodotoxin (TTX) and tetraethylammonium (TEA). No detectable changes in selectivity or instantaneous conductance were observed. No internal receptors for TTX affecting the Na conductance were found but a TEA receptor is exposed by the protein hydrolysis. 5. TEA derivatives (triethylammonium, TEA-, with an aliphatic chain, Cn) induce a partial block of the steady-state sodium current and induce a time-dependent blockage of the conductance. 6. The first effect of TEA-Cn could be described in terms of a unimolecular reaction with the following equilibrium constants: 50, 2-5, 1-0, 0-4 and 0-025 mM for TEA-C2, TEA-C4, TEA-C5, TEA-C7 and TEA-C9 respectively. 7. From the dependence of the equilibrium dissociation constant on the length of the alkyl chain we estimated the free-energy change in 560 cal/mole of CH2. The gain in free energy per CH2 group transferred from aqueous medium to the interior of a non-polar medium is 1000 cal. 8. Although with the data at hand it is impossible to propose the amino-acid sequence of the site cleaved by alkaline proteinase b, we propose that an important functional component is arginine (or lysine)