Membrane Excitability: Made Temperature-Dependent by Mutations

Abstract

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 ...

A mutant of Paramecium with increased relative resting potassium permeability

“…6) were normal. Thus, unlike the pawn mutant previously characterized (Kung and Eckert, 1972;Satow, Chang, and Kung, 1974) the mechanisms for the generation of action potential are not affected by the fast-2 mutation. Figure 7 shows that the mutation-related increase of K-permeability does not increase the K+-efflux significantly enough to affect the up-stroke of the action potential in a solution of 4 mM [Klo.…”

“…They postulated that Ba++ not only carried the action current but also blocked a K channel to reduce the leakage current which short-circuited the action current. All-or-none Ba++ action potentials were recorded in P, aurelia (Kung and Eckert, 1972;Satow, Chang, and Kung, 1974). Ba++ depolarizes paramecium membranes probably through a reduction of K+ permeability.…”

Mutants with reduced Ca activation inParamecium aurelia

“…We have previously recorded the spontaneous action potential of the ts-pawns in Ba solution (Satow, Chang & Kung, 1974). Fig.…”

“…A double mutant bred to contain both mutations, pwA1 and pwC and fails to generate avoiding reactions when grown at 23 ~ (Chang & Kung, 1973b;Satow, Chang & Kung, 1974). The evoked response was recorded from the pwA/pwC double mutant.…”

“…These are the temperature-sensitive pawns (tspawns) (Chang & Kung, 1973a, b). We have surveyed the defects of two ts-pawns grown at different temperatures and have shown that the ability to generate spontaneous Ba + § action potential remains when they are grown at room temperature but is lost at high temperatures (Satow, Chang & Kung, 1974).…”