Our results show that isovaleric acid is ananes the ticintad poles. Methylmalonic and propionic acid have anesthetic-sparing effects in tadpoles. All three compounds show a concentration-dependent enhancement of homomeric α1
glycine receptor function at concentrations that are observed in disease (up to 5mM ) (27
) as well as at the higher concentrations we explored because of the anesthetic potential of these compounds. Minimal effects were observed on α1
To determine whether the allosteric effects were similar to those of volatile anesthetics, we applied the organic acids to mutant α1
glycine and α1
receptors that were less sensitive to the modulatory effect of isoflurane and ethanol than wild type receptors (3
). With the exception of propionic acid, which had a decreased modulatory effect on the glycine receptor that was not statistically significant, all of these compounds had a smaller effect on the mutant compared to the wild type channels. This is consistent with the hypothesized shared mechanism of action with isoflurane and ethanol.
We conjectured that the organic acids exerted their anesthetic effects indirectly, by modulating lipid properties. Measurement of surface pressures of lipid monolayers has been performed for over a century (29
). We tested whether these compounds affected lipid properties by measuring pressure-area isotherms of DPPC monolayers with and without added organic acid. We used monolayers because their physical chemistry has been extensively studied (19
), the composition of the monolayer can be controlled, and its properties varied (in particular the mean area per molecule). For the pure DPPC monolayer, the shape of the isotherm can be explained as follows (). At high mean molecular areas DPPC molecules interact weakly, forming a so-called gaseous phase. With compression, the molecules eventually interact to form a liquid expanded phase, at which point surface pressure rises. Further compression leads to the formation of a second, rigid, liquid condensed phase. The appearance of this second phase leads to the loss of a degree of freedom (a consequence of Gibb’s phase rule), with the result that surface pressure becomes constant in the two phase coexistence region and a plateau is observed. Continued compression produces a monolayer that is entirely in the liquid condensed phase. This rigid phase is relatively incompressible, and surface pressures rise rapidly. Further compression eventually leads to collapse of the monolayer. A molecular dynamic simulation shows the structure of the monolayer at various points on the isotherm (20
Fig 4 Isotherm for pure DPPC monolayer. Surface pressure (total lateral pressure) is expressed in units of milliNewtons/meter (mN/m) on the y-axis. Mean molecular area is in units of Å2 on the x-axis. The monolayer is initially compressed from high (more ...)
Addition of each of the organic acids to the water subphase shifted the isotherm to higher pressures, and changed the shape of the isotherm. Increases in surface pressure of monolayers have been reported for volatile anesthetics (32
). Surfactants can also shift DPPC pressure-area isotherms (35
). In this study, the shift to higher surface pressures was most pronounced for methylmalonic acid, and least for propionic acid. In all cases these effects on the isotherms result from incorporation of the acids into the monolayers, which also changes the phase behavior of the DPPC. Of particular relevance, the acid was incorporated into the lipid monolayers at mean molecular areas characteristic of bilayers () of 60 A2
). Only at very low mean molecular areas (high surface pressures) were the acids squeezed out of the monolayer, with the isotherms approaching that of a pure DPPC monolayer.
That the organic acids interact with lipids does not rule out the possibility that these compounds may modulate ion channel function by binding directly to the channel protein. It does, however, open the door to a lipid-mediated mechanism for some of their clinical effects. The results are consistent with, but do not prove, that membrane stresses can account for the effect of the organic acids on receptor function.
Although the organic acids produce anesthetic or anesthetic-sparing effects in tadpoles, we do not know the concentrations in the tadpole that produce this effect. Our results only show that these compounds have anesthetic effects. Because we expected the animals would rapidly metabolize the organic acids, these drugs were introduced into aquarium water, which provided a large reservoir for the drug. Since only the unionized form of the acid should cross epithelial barriers, this acid is only a small fraction of the total concentration of the metabolite, and any metabolite entering the tadpole would be quickly metabolized, we expect that the concentrations of organic acid in the tadpoles are much less than the concentration we needed to apply in the aquarium.
Like ammonia (2
) and beta hydroxybutyric acid (5
), isovaleric acid, methylmalonic acid, and propionic acid are all endogenous compounds. Our results show that all are reversible central nervous system depressants. Why would an organism respond to an endogenous compound in this manner? Responding to an endogenous compound with central nervous system depression should be harmful and, hence, selected against. Yet, this does not seem to have happened. We have speculated that the basic mechanism accounting for this response may have arisen in one-celled organisms responding to interfacially active compounds in the environment (4
). The observation that inhaled anesthetics (and surfactants (3
)) enhance currents through inhibitory receptors, and inhibit currents through excitatory receptors, seems an odd physiologic response for a neuron. However, for a one-celled organism, this response makes sense: only this pattern of actions on ion channels will protect a one-celled organism’s transmembrane potential, by limiting entry of positive charges into the cell, in response to interfacially active compounds which are ubiquitous in the environment and which can modulate channel function through their effect on bilayer properties. By responding in this manner, anesthetic-sensitive ion channel architectures established in microbes could be passed on, ultimately to their descendants in multicellular organisms. It has been suggested that there is also ongoing selection for this response in animals (36
We have identified twelve new modulators of glycine receptor function over the past two years. This was accomplished primarily using as guides the known interfacial activity of surfactants (37
) and their effects on monolayer properties (35
), the predicted effect of neurotransmitters on bilayer properties (36
), molecular dynamic simulations (38
) and radioligand binding studies (39
) which show that various amino acids adsorb onto membranes. These modulators include two anionic surfactants (3
), two cationic surfactants (3
), one zwiterionic surfactant (3
), a plasticizer (40
), a neurotransmitter (GABA) (41
), and five metabolites including the three organic acids reported here (ammonia (2
), beta hydroxybutyric acid (5
), isovaleric acid, methylmalonic acid, and propionic acid). We expect that several other small, interfacially active, endogenous molecules elevated in metabolic diseases may also be allosteric modulators of ion channel function.
In summary, we have shown that isovaleric acid, methylmalonic acid, and propionic acid have anesthetic effects in animals, positively modulate glycine receptor function, and affect pressure-area isotherms of DPPC lipid monolayers. These observations may provide mechanistic insight into the central nervous system depression observed in organic acidemias, and identify new structures with anesthetic properties.