Junctional membrane permeability

Summary Substitution of extracellular Na⁺ by Li⁺ causes depression of junctional membrane permeability inChironomus salivary gland cells; within 3 hr, permeability falls to so low a level that neither fluorescein nor the smaller inorganic ions any longer traverse the junctional membrane in detectable amounts (uncoupling). The effect is Li-specific: if choline⁺ is the Na⁺ substitute, coupling is unchanged. The Li-produced uncoupling is not reversed by restitution of Na⁺. Long-term exposure (>1 hr) of the cells to Ca, Mg-free medium leads also to uncoupling. This uncoupling is fully reversible by early restitution of Ca⁺⁺ or Mg⁺⁺. Coupling is maintained in the presence of either Ca⁺⁺ or Mg⁺⁺, so long as the total divalent concentration is about 12mm. The uncoupling in Ca, Mg-free medium ensues regardless of whether the main monovalent cation is Na, Li or choline.The uncouplings are accompanied by cell depolarization. Repolarization of the cells by inward current causes restoration of coupling; the junctional conductance rises again to its normal level. The effect was shown for Li-produced uncoupling, for uncoupling by prolonged absence of external Ca⁺⁺ and Mg⁺⁺, and for uncoupling produced by dinitrophenol. In all cases, the recoupling has the same features: (1) it develops rapidly upon application of the polarizing current; (2) it is cumulative; (3) it is transient, but outlasts the current; and (4) it appears not to depend on the particular ions carrying the current from the electrodes to the cell. The recoupling is due to repolarization of nonjunctional cell membrane; recoupling can be produced at zero net currernt through the junctional membrane. Recoupling takes place also as a result of chemically produced repolarization; restoration of theK gradients in uncoupled cells causes partial recoupling during the repolarization phase.An explanation of the results on coupling is proposed in terms of known mechanisms of regulation of Ca⁺⁺ flux in cells. The uncouplings are explained by actions raising the Ca⁺⁺ level in the cytoplasmic environment of the junctional membranes; the recoupling is explained by actions lowering this Ca⁺⁺ level.     

Ionic channels through the axon membrane (a review)

Ionic channels are discrete sites at which the passive movement of ions takes place during nervous excitation. Three types of channels are distinguished. 1. Leakage channels that are permanently open to various cations. 2. Na channels that open promptly on depolarization but slowly close again (inactivate) on sustained depolarization and that are predominantly permeable to Na⁺ ions. 3. K channels that on depolarization open after some delay but stay open and that are mainly passed by K⁺ ions. The selectivity sequence of the Na channels of the squid axon (or frog nerve) is as follows: Na⁺ ≈ Li⁺>(T1+)>NH⁺ ₄?K⁺> Rb⁺, Cs⁺; that of K channels is: (T1+)>K⁺>Rb⁺>NH⁺ ₄?Na⁺, Cs⁺, Na channels are selectively blocked by tetrodotoxin (TTX) or saxitoxin (STX), K channels by tetraethylammonium ions (TEA). Either channel type is reversibly blocked when one drug molecule binds to one site per channel, the equilibrium dissociation constant of these reactions being about 3×10^?9 MTTX (or STX) and 4×10^?4 M TEA, respectively. Because of their specificity and high affinity, TTX and STX are used to “titrate” the Na channels whose density appears to be of the order of 100/Μm². The “gates” of the channels operate as a function of potential and time but independent of the permeating ion species. Drugs (e.g. veratridine) and enzymes (e.g. pronase, applied intraaxonally) cause profound changes in the gating function of the Na channels without influencing their selectivity. This points to separate structures for gating and ion discrimination. The latter is thought to be, in part, brought about by a “selectivity filter” of which detailed structural ideas exist. Recent experiments suggest that the gates of the Na channels are controlled by charged particles moving within the membrane under the influence of the electrical field.     

ACTIVATION IN VITRO OF RAT LIVER POLYRIBOSOMES

The increase in the incorporation of amino acids into protein in vitro by preparations obtained from protein-fed rats as compared with preparations obtained from carbohydrate-fed rats has been described previously. After molecular sieving through Sephadex G-25 of cell-free preparations, the difference in incorporating activity between the two types of rats was diminished in systems containing ATP, phosphoenolpyruvate, pyruvate kinase, GTP, and a mixture of amino acids. When, after molecular sieving, a mitochondrial (15,000 g) supernatant was incubated for 4 min at 35°C the polysomal pattern of the preparations was unchanged. In the presence of ATP, phosphoenolpyruvate, and pyruvate kinase the polysomal incorporating activity was low and the polysomal pattern was only slightly changed. Addition of GTP increased the activity markedly, and a more pronounced activity was observed when a mixture of amino acids was added as well. As the amino acid incorporation ability increased, monosomes were formed from the polyribosomes. The activity of the polyribosomes was severalfold higher than that of non-Sephadex-treated preparations, indicating an activation of polysomal aggregates which under the usually applied conditions of incubation and prior to molecular sieving show little or insignificant activity. It was possible to activate polyribosomes from carbohydrate-fed and protein-fed rats to almost the same extent.     

Inactivation of a newly transferred gene in recombination-deficient recipients of Escherichia coli

Summary The expression of a newly transferred lacZ ⁺ gene in lacZ recipients carrying various mutations in the recA and recB genes was studied by measuring the rates of induced synthesis of -galactosidase in zygotes formed after mating with either F or Hfr donors. The ability to synthesize -galactosidase decreases with time in both recA and recB zygotes when the lacZ ⁺ gene is transferred from an Hfr donor, but not when the lacZ gene is transferred from an F donor. There is no such inactivation of the newly transferred lacZ ⁺ gene in Rec⁺ zygotes. We conclude that the functioning of the transferred DNA is progressively inactivated in rec recipients unless the DNA is contained in an episome such as F.