Employing scan-SCAM to identify residues that compose transmembrane helix 1 (TM1) of EnvZ

The Escherichia coli sensor kinase EnvZ modulates porin expression in response to various stimuli including intracellular osmolarity, intracellular pH and periplasmic interaction with MzrA. The expression of two major outer membrane porins, OmpF and OmpC, are regulated by EnvZ, and act as passive diffusion-limited pores allowing compounds, including certain classes of antibiotics such as β-lactams and fluoroquinolones, to enter the bacterial cell. Even though allosteric processing occurs within both the periplasmic and cytoplasmic domains of EnvZ, how the transmembrane domain bi-directionally transmits these signals remains not fully understood. Here, we employ a library of single-Cys-containing EnvZ proteins to perform scan-SCAM in order to map the precise residue composition of TM1. Our results demonstrate that residue positions 19 through 30 reside within the membrane core and compose a tightly packed portion of TM1. We also show that positions 15 through 18 and position 31 are interfacial and slightly splayed apart compared to those tightly packed within the hydrophobic core. Finally, we reveal that residue positions 33 and 34 reside in the periplasm and participate in robust protein-protein interactions, while the periplasmic positions 35 through 41 exhibit helical periodicity. We conclude by synthesizing these new insights with recent high-resolution structural information into a model of membrane-spanning allosteric coupling between the periplasmic and cytoplasmic domains of EnvZ.

Potassium homeostasis

The equilibrium between the concentration of K+ in the extracellular space (low) and the intracellular compartment (high) is crucial for maintaining the electrical properties of excitable and non-excitable cells, because it determines the membrane resting potential. The high intracellular concentration of K+ (120–140 mmol/L) also contributes to the intracellular osmolarity, a determinant of cell volume. It is therefore crucial to finely tune both extracellular and intracellular K+ concentrations. There is a coordinated regulation between processes/mechanisms that store/release K+ from internal stores (internal balance) and those that retain/excrete K+ (external balance).

Adjustment of osmotic pressure coupled with change of growth mode in Spirogyra

Spirogyra living in running water forms a rhizoid which anchors it to the substratum. Rhizoid differentiation can be induced in the laboratory by severing algal filaments. The terminal cell changes the growth mode from diffuse growth to tip growth, and finally differentiates to be a rhizoid. We found that the intracellular osmolarity of the rhizoid was significantly lower than that of other interjacent cells which did not form rhizoids. The decrease in the intracellular osmolarity began before the start of tip growth. TEA, a K+ channel blocker, inhibited the decrease in the intracellular osmolarity of the terminal cells; increase in the external K+ also inhibited this. It was suggested that K+ efflux through K+ channel is involved in the adjustment of osmotic pressure. When the adjustment of osmotic pressure was inhibited, tip growth did not start, inevitably, no rhizoid was formed. In Spirogyra sp. which was unable to form rhizoids, the terminal cell did not show the adjustment of osmotic pressure. Thus, this adjustment seems to be intimately coupled with the rhizoid differentiation. Possible roles of the adjustment of osmotic pressure in rhizoid differentiation are discussed.

Activation of the Protein Kinase C1 Pathway upon Continuous Heat Stress in Saccharomyces cerevisiae Is Triggered by an Intracellular Increase in Osmolarity due to Trehalose Accumulation

ABSTRACT This paper reports on physiological and molecular responses of Saccharomyces cerevisiae to heat stress conditions. We observed that within a very narrow range of culture temperatures, a shift from exponential growth to growth arrest and ultimately to cell death occurred. A detailed analysis was carried out of the accumulation of trehalose and the activation of the protein kinase C1 (PKC1) (cell integrity) pathway in both glucose- and ethanol-grown cells upon temperature upshifts within this narrow range of growth temperatures. It was observed that the PKC1 pathway was hardly activated in a tps1 mutant that is unable to accumulate any trehalose. Furthermore, it was observed that an increase of the extracellular osmolarity during a continuous heat stress prevented the activation of the pathway. The results of these analyses support our hypothesis that under heat stress conditions the activation of the PKC1 pathway is triggered by an increase in intracellular osmolarity, due to the accumulation of trehalose, rather than by the increase in temperature as such.

Regulation of Cellular Osmolarity and Volume in Tetrahymena

1. Tetrahymena pyriformis are hyperosmotic to external media of osmolarities from 2 to 171 m-osmole/l. The intracellular osmolarity, determined by freezing-point depression, is 111 m-osmole/kg cells in dilute media, and increases linearly with increasing external osmolarities. 2. Over 80% of the intracellular osmolarity can be attributed to the concentration of sodium, potassium, chloride and the free amino acids. 3. In response to an increase in the external osmolarity, Tetrahymena regulates its intracellular osmolarity by increasing the concentrations of free amino acids. 4. The regulation of cellular volume under conditions of osmotic stress is achieved by an increase in the amount of osmotically active solutes and the regulation of the rate of elimination of fluid by the contractile vacuole.

Extracellular and Metabolic Factors Affecting the Efflux and Influx of Erythrocyte Water

Water exchanges between rabbit erythrocytes and extracellular solutions equidistant from intracellular osmolarity were studied by freezing point depression techniques. Water efflux was always less than water influx and both were hypotonic to the intracellular and extracellular fluids. The magnitudes of these water exchanges were not dependent on the presence of extracellular cation. Stimulation of oxygen uptake by the addition of glucose and methylene blue increased water influx and, possibly, decreased water efflux. This could not be accounted for by accumulation of osmotically active intracellular metabolic products. O2 uptake was markedly decreased during cellular dehydration, was slightly decreased during cellular overhydration, and was maximum at the water content of erythrocytes when suspended in a medium isotonic with plasma.