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Sodium ion transport across tight epithelia has been investigated particularly extensively by studying two model systems: the urinary bladder of the toad and the frog skin. The greatest advantage presented by these models is the capability of monitoring net transepithelial Na+ flux simply, precisely, and instantaneously by measurement of the short circuit current (ISC). Many of the caveats involved in the measurement are discussed in detail. In order to fully characterize the forces driving Na+ movement across the series apical and basolateral membranes, it is necessary to measure intracellular potential and ionic composition. Such measurements are far more easily conducted with frog skin than with toad bladder, using the major biophysical techniques currently available. Regulation of transepithelial Na+ movement across tight epithelia is largely conducted at the apical membranes. This regulation can be clarified by study of the isolated Na+ channels in membrane vesicles. Such vesicles are far more easily prepared from toad urinary bladder than from frog skin. The strengths and potential misappropriations of this technique are considered in detail.

Our previous experiments in membranes prepared from rat heart and brain led us to suggest that the binding of agonists to the muscarinic receptors and to the Na+ channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [3H]acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [3H]batrachotoxin to Na+ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22Na+ uptake in the presence and absence of tetrodotoxin, which blocks Na+ channels. Our findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na+ channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components--receptor, G-protein, and Na+ channel--is such that at resting potential the muscarinic receptor induces opening of Na+ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.

Photoacoustic (PA) measurements provide, by the very nature of the PA effect, the possibility to obtain information on the optical and thermal properties of samples. In addition they can yield information on the enthalpy changes and characteristic times involved in photo-induced processes as the acoustic signal is proportional to the hear produced following the absorption of the modulated excitation. In the study of optical properties the relative insensitivity to scattered light of the PA signal makes such measurement an attractive way to measure biological samples in vivo, or, at least, without the need to isolate the absorbing compounds. The dependence of the PA signal on the thermal properties of the sample is particularly useful when heterogeneous samples are studied. As a photocalorimetric method the technique shows considerable promise in the study of photo-bioenergetics, especially photosynthesis. Only in special cases can analytical applications of the PA method compete with fluorescence measurements for detection, and with increasingly sophisticated optical transmission and reflectance techniques (for identification).. However, the PA method may find important uses in fundamental research and in applied areas such as biomedicine and agricultural biochemistry.

The procedure we have described provides a simple, convenient, and sensitive method to assay conductive ion fluxes in membrane vesicles. It is particularly useful for detecting channels in heterogeneous populations of vesicles. The principal advantages are similar to those of sensitive enzyme assays, namely, screening for existence of channels in different membrane fractions, assaying purified channel proteins, large-scale testing of pharmacological agents, antibodies, etc. and in studies of macroscopic regulatory features, including channel activity or density in different states and interaction with regulatory ligands. In the future one can expect further applications in detecting synthesis of channel proteins, gene expression, etc. The tracer assay does not provide much information on molecular characteristics such as single-channel conductance, voltage sensitivity, and ion specificity. It therefore serves other purposes to those of the modern biophysical methods such as patch-clamp, noise analysis, and study of channels incorporated into bilayers.

Inside-out cell envelopes from Halobacterium halobium R1M1 have been isolated by sonication and subsequent sucrose-density-gradient fractionation. The inverted cell envelopes transport protons from the exterior of the vesicles by a light-dependent, uncoupler-inhibited process. Enzymes usually facing the inside of the cell are exposed to the external medium in this preparation.

The kinetics of light-induced acidification and of the subsequent dark-induced alkalization in suspensions of sub-bacterial particles of Halobacterium halobium may be expressed as the sum of two exponentials, indicating two processes (Eisenbach, M., Bakker, E.P., Korenstein, R. and Caplan, S.R. (1976) FEBS Lett. 71, 228--232). We studied the effects of carbonyl cyanide p-trifluoromethyoxy phenyl-hydrazone, nigericin, gramicidin D, valinomycin, and monactin on the extents and the rate constants of the two processes. The various ionophores affected the two processes differently and in general the slower process was more sensitive to their presence. Valinomycin and monactin had relatively minor effects, apparently due to the high ionic strength of the suspension. When an artificial membrane potential was created in the dark, the light-induced acidification was preceded by a transient alkalization as is usually observed in intact cells. These results are discussed in the light of a suggested model accounting for the two processes (Caplan, S.R., Eisenbach, M., Cooper, S., Garty, H., Klemperer, G. and Bakker, E.P. (1977) in Bioenergetics of Membranes (Packer, L., Papageorgiou, G.C. and Trebst, A., eds.), pp. 101--114, Elsevier/North-Holland Biomedical Press, Amsterdam), taking into account the different selectivities of the ionophores applied.

The polar lipids of the purple membrane were exchanged for different phosphatidylcholine species. The resulting complexes had the same protein to lipid-phosphorus ratio as the natural membrane, but only about 0.5-1.0 mole of original lipid was still present per mole of bacteriorhodopsin. In such complexes the bacteriorhodopsin photocycle is slowed down 10-20 times, but the strong protein-protein interaction is not abolished. Due to the slow rate of the photocycle we were able to measure in the light the ratio between net proton release and net accumulation of the last intermediate of the photocycle, the unprotonated M412. This ratio was not constant and equal to 1.0, as expected for a single deprotonation reaction, but varied with pH from 1.5 to 0.4. The variable ratio suggests that light-induced conformational changes occur in the nonchromophore part of the protein, which shift the pKa values of unidentified groups so as to cause binding or release of additional protons. A similar conclusion was drawn from experiments on the kinetics of proton transfer by bacteriorhodopsin in subbacterial particles of Halobacterium halobium and in reconstituted bacteriorhodopsin proteoliposomes. However, in this case light-induced association and dissociation of additional protons occurs simultaneously on different sides of the membrane.

Light-induced Na+ efflux was observed in sub-bacterial particles of Halobacterium halobium loaded and suspended in 4 M NaCl solution. The Na+ efflux was not ATP driven, since ATPase inhibitors were without effect or even enhanced efflux at low light intensity. Uncouplers, on the other hand, inhibited Na+ efflux, the inhibition being complete at low light intensity. The Na+ efflux was accompanied by proton influx. Both processes were dependent on light intensity, unaffected or enhanced by ATPase inhibitors and similarly affected by uncouplers. Proton influx was not observed in particles loaded with 4 M KCl instead of 4 M NaCl. Na+ transport in the dark could be induced by artificial formation of a pH difference across the membrane; changing the sign of the pH difference reversed the direction of the Na+ transport. Proton influx in the dark followed the artificial formation of a sodium gradient [Na+]in less than [Na+]out). These results may be explained by a Na+/H+ antiport mechanism. The fluxes of Na+ and H+ were of comparable magnitude, but the initial rate of Cl- efflux in the same experiment was one-third of the initial rate of Na+ efflux. Consequently Cl- is not regarded as a participant in the Na+ efflux mechanism.

Ouabain-blocked toad urinary bladders were maintained in Na+-free mucosal solutions, and a depolarizing solution of high K+ activity containing only 5 mM Na+ on the serosal side. Exposure to mucosal sodium (20 mM activity) evoked a transient amiloride-blockable inward current, which decayed to near zero within one hour. The apical sodium conductance increased in the initial phase of the current decay and decreased in the second phase. The conductance decrease required Ca2+ to be present on the serosal side and was more rapid when the mucosal Na+ activity was higher. At 20 mM mucosal Na+ and 3 mM serosal Ca2+ the initial (maximal) rate of inhibition amounted to 20% in 10 min. The conductance decrease could be accelerated by raising the serosal Ca2+ activity to 10 mM. The inhibition reversed on lowering the serosal Ca2+ to 3 microM and, in addition, the mucosal Na+ to zero. Exposure of the mucosal surface to the ionophore nystatin abolished the Ca2+ sensitivity of the transcellular conductance, showing that the Ca2+-sensitive conductance resides in the apical membrane. The data imply that in the K+-depolarized epithelia, cellular Ca2+, taken up from the serosal medium by means of a Na+-Ca2+ antiport, cause feedback inhibition by blockage of apical Na+ channels. However, the rate of inhibition is small, such that this regulatory mechanism will have little effect at 1 mM serosal Ca2+ and less than 20 mM cellular Na+.

The uptake of rubidium in intact Halobacterium halobium cells was followed, and found to be light-dependent. The exchange process is slow, the steady-state rate of 86Rb+/Rb+ exchange being given by k. = 6.3 - 10(-4) min-1. Starved cells exhibited a faster rate than unstarved cells. The influx of 86Rb+ was almost completely blocked in the presence of proton conductors (CCCP, FCCP, and SF 6847), and was sensitive to the presence of the permeant cation TPMP+. Valinomycin very slightly increased the rate of uptake, while 1 - 10(-6) M nigericin showed significant inhibition. On the other hand, release of 86Rb+ was not light-dependent, although still affected by uncouplers, TPMP+, and nigericin. These experimental observations may be explained in terms of a passive flux driven by an electrical potential difference, and influenced by positive isotope interaction within the membrane. In carefully matched influx-efflux studies, the extent of the positive isotope interaction was measured. Using the formal treatment of Kedem and Essig, the ratio (exchange resistance)/(resistance to net flow) for 86Rb+ was found to be 1.7.