Calcium block of Na+ channels and its effect on closing rate

Calcium ion transiently blocks Na+ channels, and it shortens the time course for closing of their activation gates. We examined the relation between block and closing kinetics by using the Na+ channels natively expressed in GH3 cells, a clonal line of rat pituitary cells. To simplify analysis, inactivation of the Na+ channels was destroyed by including papain in the internal medium. All divalent cations tested, and trivalent La3+, blocked a progressively larger fraction of the channels as their concentration increased, and they accelerated the closing of the Na+ channel activation gate. For calcium, the most extensively studied cation, there is an approximately linear relation between the fraction of the channels that are calcium-blocked and the closing rate. Extrapolation of the data to very low calcium suggests that closing rate is near zero when there is no block. Analysis shows that, almost with certainty, the channels can close when occupied by calcium. The analysis further suggests that the channels close preferentially or exclusively from the calcium-blocked state.

Cell membrane fluctuations are regulated by medium macroviscosity: Evidence for a metabolic driving force

Extracellular fluid macroviscosity (EFM), modified by macromolecular cosolvents as occurs in body fluids, has been shown to affect cell membrane protein activities but not isolated proteins. In search for the mechanism of this phenomenon, we examined the effect of EFM on mechanical fluctuations of the cell membrane of human erythrocytes. The macroviscosity of the external medium was varied by adding to it various macromolecules [dextrans (70, 500, and 2,000 kDa), polyethylene glycol (20 kDa), and carboxymethyl-cellulose (100 kDa)], which differ in size, chemical nature, and in their capacity to increase fluid viscosity. The parameters of cell membrane fluctuations (maximal amplitude and half-width of amplitude distribution) were diminished with the elevation of solvent macroviscosity, regardless of the cosolvent used to increase EFM. Because thermally driven membrane fluctuations cannot be damped by elevation of EFM, the existence of a metabolic driving force is suggested. This is supported by the finding that in ATP-depleted red blood cells elevation of EMF did not affect cell membrane fluctuations. This study demonstrates that (i) EFM is a regulator of membrane dynamics, providing a possible mechanism by which EFM affects cell membrane activities; and (ii) cell membrane fluctuations are driven by a metabolic driving force in addition to the thermal one.

The effect of protein concentration on ion binding.

The concentration of protein in a solution has been found to have a significant effect on ion binding affinity. It is well known that an increase in ionic strength of the solvent medium by addition of salt modulates the ion-binding affinity of a charged protein due to electrostatic screening. In recent Monte Carlo simulations, a similar screening has been detected to arise from an increase in the concentration of the protein itself. Experimental results are presented here that verify the theoretical predictions; high concentrations of the negatively charged proteins calbindin D9k and calmodulin are found to reduce their affinity for divalent cations. The Ca(2+)-binding constant of the C-terminal site in the Asn-56 --> Ala mutant of calbindin D9k has been measured at seven different protein concentrations ranging from 27 microM to 7.35 mM by using 1H NMR. A 94% reduction in affinity is observed when going from the lowest to the highest protein concentration. For calmodulin, we have measured the average Mg(2+)-binding constant of sites I and II at 0.325, 1.08, and 3.25 mM protein and find a 13-fold difference between the two extremes. Monte Carlo calculations have been performed for the two cases described above to provide a direct comparison of the experimental and simulated effects of protein concentration on metal ion affinities. The overall agreement between theory and experiment is good. The results have important implications for all biological systems involving interactions between charged species.

Cancer vaccines: the interleukin 2 dosage effect.

Cancer vaccines genetically engineered to produce interleukin 2 have been investigated intensively in a series of animal models and are at the point of entering into clinical trials. In this study we demonstrate a strong correlation between the rate of interleukin 2 production and the protection efficiency of murine S91 melanoma cell (clone M-3) vaccines. Best immunization is achieved with vaccines producing medium interleukin 2 levels of 1000-3000 units per 10(5) cells per day. Reduced interleukin 2 production evokes a corresponding decline in the number of successfully treated animals. Unexpectedly, when interleukin 2 expression is raised to high levels of 5000-7500 units per 10(5) cells per day, protection is completely absent because of impaired generation of tumor-specific cytotoxic T lymphocytes. In comparison, granulocyte-macrophage colony-stimulating factor as immunomodulator induces substantial immunization even at a moderate level of secretion and protects all animals at the maximal obtainable level of secretion. Our findings demonstrate the importance of the interleukin 2 level produced by genetically modified tumor cells and may have substantial impact for the clinical application of cancer vaccines.