Role of concentration and size of intracellular macromolecules in cell volume regulation

Abstract

To gain insight into the mechanism(s) by which cells sense volume changes, specific predictions of the macromolecular crowding theory (A. P. Minton. In: Cellular and Molecular Physiology of Cell Volume Regulation, edited by K. Strange. Boca Raton, FL: CRC, 1994, p. 181-190. A. P. Minton, C. C. Colclasure, and J. C. Parker. Proc. Natl. Acad. Sci. USA 89: 10504-10506, 1992) were tested on the volume of internally perfused barnacle muscle cells. This preparation was chosen because it allows assessment of the effect on cell volume of changes in the intracellular macromolecular concentration and size while maintaining constant the ionic strength, membrane stretch, and osmolality. The predictions tested were that isotonic replacement of large macromolecules by smaller ones should induce volume decreases proportional to the initial macromolecular concentration and size as well as to the magnitude of the concentration reduction. The experimental results were consistent with these predictions: isotonic replacement of proteins or polymers with sucrose induced volume reductions, but this effect was only observed when the replacement was > or = 25% and the particular macromolecule had an average molecular mass of < or = 20 kDa and a concentration of at least 18 mg/ml. Volume reduction was effected by a mechanism identical with that of hypotonicity-induced regulatory volume decrease, namely, activation of verapamil-sensitive Ca2+ channels.

Intramolecular and Intermolecular Fluorescence Resonance Energy Transfer in Fluorescent Protein-tagged Na-K-Cl Cotransporter (NKCC1)

“…The mechanisms by which cell volume regulate cellular processes remain for the most part elusive, and macromolecular 7 The approximate YFP levels in our constructs relative to hslo-667-YFP in the NKCC-CЈ cotransfectant (k in Fig. 5 crowding has been seen as a strong candidate for the first step in the signaling process (48,49). However, although crowding or other physical factors may directly affect NKCC1, there is also strong evidence that the activities of upstream modulatory kinases (50,51), particularly WNK (with no lysine) kinases (52)(53)(54), are determined by changes in intracellular [Cl Ϫ ] and cell volume.…”

Macromolecular Crowding as a Cell VolumeSensor

“…Subsequent experiments in dog red blood cell ghosts [2,3] and internally perfused barnacle muscle cells [4] supported this hypothesis by demonstrating that introducing natural or artificial macromolecules into these cells in vitro to alter macromolecular crowding can produce volume regulatory changes. Macromolecular crowding [5] and its possible role in cell volume regulation [6][7][8] have been extensively reviewed.…”

“…It was quickly recognized, however, that the model in its original form is difficult to reconcile with the particular conditions of the experiments in dog erythrocytes and internally perfused barnacle muscle cells [4]. The model specifies association of soluble kinase with a membrane associated transporter, but the hemolysis or perfusion was designed to alter macromolecular crowding by more or less completely replacing endogenous soluble macromolecules with those whose crowding effect was to be tested.…”

“…How could the hypothetical soluble volume sensing macromolecules not be removed at the same time? This led Minton (personal communication) to suggest alternative models [4]. The experimental findings imply that the regulator must somehow be tethered to the membrane, so that it is not washed out upon perfusion or hemolysis.…”

“…The possible role of macromolecular crowding as a signal for cell volume regulation was also tested with internally perfused barnacle muscle cells [4]. A single cell was cannulated through its cut end with a doublebarrel capillary tube (for perfusion), a current passing wire, and a voltage-sensing electrode.…”