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Transmembrane and intracellular distrubution of chloride and potassium in single striated muscle fibers of the giant barnacle Gayton, David Charles

Abstract

The initial aim of this thesis was to study the transmembrane and intracellular distribution of Cl⁻ in single striated muscle fibers of the giant barnacle, Balanus nubilus. The study was expanded to include the transmembrane distribution of K⁺ and a consideration of the Donnan relation. Chloride sensitive Ag-AgCl microelectrodes were inserted into single fibers to measure the activity of Cl⁻ in the myoplasm. The myoplasmic Cl⁻ activity was just slightly higher than predicted from the Nernst equation, indicating that Cl⁻ is probably distributed passively across the membrane of the resting fiber. Comparison of the electrode results with analytic results suggested that less than half of the fiber Cl⁻ is free in the myoplasm. Most of this extra Cl⁻ was located in an extracellular space, determined, by ¹⁴C-sorbitol penetration and a Cl⁻ washout method, to occupy about 5% of the fiber volume. Electron micrographs indicate that this space is comprised of large clefts and smaller tubules which penetrate deeply into the fiber. In a second series of experiments, the kinetics of Cl⁻ exchange and the outflow of intracellular Cl⁻ into solutions with reduced Cl⁻ concentrations were studied. The results indicate that the intracellular Cl⁻ can be divided about equally between a rapidly exchanging fraction which is free in the myoplasm and a slowly exchanging fraction which is either bound or compartmentalized. Furthermore, the free myoplasmic Cl⁻ appears to be excluded from about 45% of the intracellular water. In the third series of experiments, the activities of K⁺ and Cl⁻ in the myoplasm of single fibers at differing states of equilibrium were measured by means of ion sensitive micro-electrodes. Fibers were equilibrated in Ringer solution which was modified in three ways: [K]₀ and [C1]₀ were varied but [K]₀ x [Cl]₀ and [K]₀ + [Na]₀ kept constant; [K]₀ was varied but [Cl]₀ and [K]₀ + [Na]₀ kept constant; [K]₀, [Cl]₀, and osmolarity were increased. The results provide rigorous proof for the widely held theory that, under most equilibrium conditions, K⁺ and Cl⁻ are distributed across the membrane of a muscle fiber according to the Donnan relation. The results also confirm the finding.that Cl⁻ is apparently excluded from 45% of the intracellular water and earlier findings from this laboratory that K⁺ is excluded from a smaller fraction of the intracellular water. Finally, two experiments are described which show that the intracellular distribution of K⁺ and Na⁺ is not the same in fibers from barnacles collected in two different locations. It is suggested that although the sum of the K⁺ plus Na⁺ that is bound or compartmentalized may be constant, the differences in the results could be due to an inverse variation in the amount of each cation that is bound or compartmentalized. The results of this study are in accord with recent findings in this and other laboratories which indicate that monovalent inorganic ions and water are distributed in a heterogeneous manner in the striated muscle fiber. The intracellular Cl⁻of the barnacle muscle that is not free in the myoplasm must be either bound to proteins or accumulated in the sarcoplasmic reticulum; at present there is not sufficient evidence to select one or the other (or both) of these possibilities. It is proposed that the free myoplasmic Cl⁻ is excluded from water that is ordered by the cell proteins and partially excluded from water that lies within the electrical field that surrounds the negatively charged proteins.

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