Two heat-sensitive "pawn" mutants of Paramecium aurelia are capable of avoiding reactions when grown at 23 degrees C but not at 35 degrees C. Electrophysiological analyses show that Ca activation is reduces in the mutants even when they are grown at 23 degrees C. The maximal rate of rise and the peak of the evoked action potential (Ca-spike) in the mutants are smaller than those of wild type in a K-solution. After suppression of K conductance by either TEA+ or Ba++, the action potentials of the mutants peak at the same level as that of wild type. However, the maximal rate of rise of the mutants remains only about half that of wild type. Thus, the mutations affect Ca activation but not K activation. Incubation at a high temperature (35 degrees C) further reduces Ca activation to almost zero in the mutants but has little or no effect on wild type. This almost complete loss of Ca activation explains the lack of avoiding reactions when the mutants are grown at high temperatures. A double mutant containing two heat-sensitive mutations shows extremely reduced Ca activation even when grown at 23 degrees C.
Membrane characteristics of neuron somata in the medulla terminalis ganglionic X-organ of crayfish have been investigated with intracellular glass microelectrodes. The soma membrane developed action potentials with 10-20 mv of overshoot. Delayed rectification appeared at 10-20 mv above resting membrane potential. In 50% of the neuron somata examined, action potentials were observed in Na-free medium or TTX medium. The peak potential level of the spike in these media depended on the extracellular concentration of Ca ion. It increased with the Ca concentration. In low calcium media, the peak potential level of the spike varied with Na concentration. Action potentials of the X-organ-sinus gland tract disappeared after bathing in Na-free or TTX medium, suggesting that the conductive action potential was dependent on Na ions. From these results, it is concluded that there are two systems in the neuron soma, one of which responds to the Na ion and the other, to the Ca ion. Inhibitory innervation of the X-organ by the cerebral ganglion was manifested by IPSP's when the optic peduncle was stimulated. A postulated connection between the Ca-dependent spike and the release of hormone in X-organ neuron somata is discussed.
Fast-2, a membrane mutant of Paramecium aurelia, is due to a single-gene mutation and has behavioral abnormalities. Intracellular recordings through changes of external solutions were made. The mutant membrane hyperpolarized when it encountered solutions with low K+ concentration. This hyperpolarization and other associated activities were best observed in Ca- or Na-solutions devoid of K+. Membrane potential was plotted against the concentration of K+ (0.5 to 16 mM) in solutions of fixed Na+ or Ca++ concentration. The slopes of the curves for the mutant membrane were steeper than those for the wild type at the lower concentrations of K+. Inclusion of 2 mM tetraethylammonium chloride (TEA-Cl) counteracted the mutational effects. Spontaneous action potentials in Ba-solution and the electrically evoked action potentials in various solutions are normal in this mutant. We conclude that the resting permeability to K+ relative to the permeabilities to Na+ and Ca++ has been increased by the mutation.
Three mutants of Paramecium aurelia with genetic lesions at two unlinked loci lost their ability to generate action potentials when grown at high temperatures. Action potentials found at room temperature were slightly aberrant. Kinetics of phenotypic changes after temperature shifts showed that excitation is not immediately sensitive to temperature change in these mutants. The initiation of an action potential must rely on many different gene products (presumably membrane proteins) which are open to modification by conditional as well as unconditional mutations.To fathom the material basis of behavior it is essential that we provide a molecular mechanism for excitation and not merely a detailed description of the phenomenon. Membrane proteins are implicated in the voltage-sensitive ion gating processes vital to the generation of action potentials (1). However, the identity of the macromolecules and the mechanisms involved are virtually unknown. One common modern biological approach to macromolecular mechanisms is to monitor the system after inducing molecular modifications by genic mutations. Correlations between the molecular modification and altered system output can shed light on the operating principle. Such an approach has already been taken in the analyses of the behavior of motile bacteria (2), nematodes (3), and insects (4). The ciliated protozoan Paramecium aurelia is an experimental organism well suited to this approach to the problem of the membrane molecular mechanisms, because it is amenable to both genetic and electrophysiological analyses. Behavioral mutants with membrane defects have been isolated (5, 6) and the electrophysiological correlates of their behavioral aberrations have been identified in some cases (7,8). This paper shows that excitation can become temperature-dependent if the relevant gene products, presumably proteins, are made heat-sensitive by conditional mutations.Heat-sensitive mutations have been exploited in a variety of biological studies (9, 10). Heat-sensitive behavioral mutants of P. aurelia were recently isolated (6, 11) with a modification of a screening method previously devised (5, 12). Three of the five strains of heat-sensitive "Pawns" are analyzed here. Pawns are mutants that are not capable of performing the avoiding reaction in response to stimuli. The avoiding reaction in the wild type consists of a short period of reversal of the ciliary beat, causing the cell to back away from the source of mechanical, chemical or other stimulations (13). Ciliary reversal is correlated with an increase in the internal Ca++ concentration (14), probably resulting from the calcium influx associated with an action potential. This voltage-sensitive gating mechanism for Ca++ is thought to be very similar to the comparable mechanism for Na+ in 2703 Solutions. In the physiological studies, the adaptation medium was a potassium solution consisting of 4 mM KOH, 1 mM CaCl2, and 1 mM citric acid. The barium solution was 4 mM BaCl2 and 1 mM CaCl2. All solutions were buffered by 1 ...
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