Vesicle transmembrane potentials

Stimulation of active H+ extrusion from roots (Cesco, 1995 Pinton et al., 1997 Table 9.1) and transmembrane potential hyperpolarization (Slesak and Jurek, 1988) indicated the involvement of the PM H+-ATPase in the increased nutrient uptake generally observed in the presence of humic substances. Direct proof of an interaction between humic molecules and the PM H+-ATPase has been obtained by Vara-nini et al. (1993), who demonstrated that low-molecular-weight (<5kDa) humic molecules at concentrations compatible with those present in the rhizosphere can stimulate the phospho-hydrolytic activity of this enzyme in isolated PM vesicles (Table 9.1). Further proof of the action of humic molecules on PM FT-ATPase activity and on nutrient uptake mechanisms was obtained when studying the effect of these molecules on NO3 uptake. Transport of this nutrient is a substrate-inducible process and involves FT co-transport. At higher uptake rates, the levels and activity of root PM FT-ATPase increased (Santi et al., 1995). The short-term (4h) contact... 

Figure 4. Effect of kmophores (A) and transmembrane potentials (B) on quantum yield of heptylviologen reduction in the vesicle system where 4 is the quantum yield. CCCP (carbonyl cyanide m-chlorophenylhydrazone) is the H carrier, and valino-mycin is the K carrier gramicidin makes the membrane permeable for cations such...

While vesicle deformation in AC fields concerns stationary shapes, DC pulses induce short-lived shape deformations. In different studies, the pulse duration has been typically varied from several microseconds to milliseconds, while studies on cells have investigated a much wider range of pulse durations-from tens of nanoseconds to milliseconds and even seconds [80], as discussed in other chapters of this book. Various pulse profiles, unipolar or bipolar, as well as trains of pulses have been also employed (e.g., [81, 82]). Because the application of both AC flelds and DC pulses creates a transmembrane potential, vesicle deformations of similar nature are to be expected in both cases. However, the working fleld strength for DC pulses is usually higher by several orders of magnitude. Thus, the degree of deformation can be different. 

The typical decay hme for the relaxahon of nonporated vesicles, t, is of the order of 100 ps. It is set by the relaxahon of the membrane tension achieved at the end of the pulse. The membrane tension, acquired during the pulse, also referred to as electric tension, arises from the transmembrane potenhal, ( , built across the membrane during the pulse. Lipid membranes are impermeable to ions and, in the presence of an electric field, charges accumulate on both sides of the bilayer, which gives rise to this transmembrane potential [91] ... 

Above some electroporation threshold, the transmembrane potential cannot be further increased, and can even decrease due to transport of ions across the membrane [91, 95]. The phenomenon of membrane electroporation can also be understood in terms of tension. If the total membrane tension exceeds the lysis tension c ys, the vesicle ruptures. This corresponds to building up a certain critical transmembrane potential, = Pc- According to Eqs. (7.3) and (7.4), this porahon potenhal Pc depends on the inihal membrane tension Co as previously reported [59, 89, 90, 96, 97]. The crihcal hansmembrane potenhal for cell membranes is about IV (e.g., [98, 99]). 

Similar, but smaller, potentiating effects of H,-receptor stimulation on cyclic AMP responses to impromidine or adenosine have been reported in dissociated brain cells [196] and a preparation of vesicles from postsynaptic membranes of guinea-pig cerebral cortex [200-204]. This latter particulate preparation contains vesicular entities which maintain a degree of metabolic and functional integrity and have transmembrane potentials in the - 58 to - 78 mV range... 

A neuron, or nerve cell, is an example of an excitable cell. These cells have cell bodies (or soma) containing the nucleus and elongated processes called axons and dendrites. These cells form a complex web with many connections. Figure 11 shows a schematic diagram of a connection between two such nerve cells. The presynaptic neuron is separated from the postsynaptic neuron by a small gap known as the synapse, typically 2-800 A wide. Presynaptic nerve endings contain small sacs or vesicles filled with one of several compounds called neurotransmitters. The postsynaptic neuron has receptor sites for specific neurotransmitters located on the cell membrane. When appropriately stimulated, and area of a presynaptic neuron membrane becomes depolarized (the transmembrane potential becomes more positive). The depolarized area propagates down the axon very rapidly. This wave-like movement of depolarization is called the action potential. When the depolarized areas reaches the nerve ending, the vesicles move to the cell wall, fuse with it, and dump their contents into the synaptic cleft—a process called exocytosis. (Exocytosis is accepted as the mechanism of neurotransmitter release in the peripheral nervous system, but it has not yet been demonstrated... 

Komberg RD, McNamee MG, McConnell HM (1972) Measurement of transmembrane potentials in phospholipid vesicles. Proc Natl Acad Sci USA 69 1508-1513... 

Other ESR studies of membrane structure include the determination of phospholipid flip-flop rates (Kornberg and McConnell, 1971), studies of membrane permeability, measurements of vesicle internal volumes and transmembrane potentials (Marsh et aL, 1976 McNamee and McConnell, 1973). 

From experiments on planar bilayer membranes (BLM), it was known that lipid bilayers were not able to withstand an increase in the applied voltage above a threshold value. A conductive state followed by a rupture was observed for values of the order of 200 mV. Electropulsation induces a transmembrane potential modulation, bringing a similar membrane instability. Indeed experiments on pure lipid vesicles showed that upon the field pulse the lipid bilayer could become leaky. This was observed on line by the associated increase in conductance of a salt-filled vesicle suspension [26]. But larger molecules could leak out and be directly detected outside the vesicles as observed with radiolabelled sucrose [27] or fluorescent dyes [28]. A very fast detection of the induction of membrane leakage is obtained by electrical conductance and light scattering... 

The vesicle assay previously used ves a good survey of transporter activity under a fixed set of conditions. However it is not suited to exploration of transport activity as a function of the sign and magmtude of the transmembrane potential. Bilayer conductance, or single channel recording techniques are required (21). In this techmque, a lipid bilayer is formed across a small hole in a Teflon barrier, by direct application of the lipid in decane to the hole. Under favorable conditions, Ae lipid tl s to a bilayer membrane which electrically isolates the two halves of the cell. T icdly KCl is used as an electrol, and electrical contact is made via Ag/AgQ wires directly inserted into the solutions. A high impedance operational amplifier circuit (bUayer clamp) can then be used to apply a fixed transmembrane potential and to monitor the current which flows as a function of time. The two sides of the membrane are independently accessible, so different sequences of transporter addition, control of pH, and other variables are in principle j ssible. 

Early studies in our laboratory on membrane potentials and the uptake of weak bases used for the measurement of ApH led to the recognition that a variety of chemotherapeutic drugs could be accumulated within LUVs exhibiting transmembrane pH gradients (59). This remote-loading technique, so named because drug is loaded into preformed vesicles, is based on the membrane permeability of the neutral form of weakly basic drugs such... 

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