Atx1-like chaperones and their cognate P-type ATPases: copper-binding and transfer

Copper is an essential yet toxic metal ion. To satisfy cellular requirements, while, at the same time, minimizing toxicity, complex systems of copper trafficking have evolved in all cell types. The best conserved and most widely distributed of these involve Atx1-like chaperones and P_1B-type ATPase transporters. Here, we discuss current understanding of how these chaperones bind Cu(I) and transfer it to the Atx1-like N-terminal domains of their cognate transporter.     

Cell Cycle activation and ouabain-sensitive ion movements of 3T3 and C3H-10T½ fibroblasts

The introduction of either PGF_2α (10^?7 M) or TPA (10^?7 M) stimulated, ouabain-sensitive ⁸⁶Rb⁺ influx at 30 min in postconfluent 3T3-4 mouse fibroblast cultures by 117% and 124%, respectively. Both TPA and PGF_2α at these concentrations stimulated the incorporation of ³H-TdR into DNA. TPA had the greatest stimulatory effect, which was similar to that obtained with 10% fetal calf serum. In accord with the idea that modulation of membrane processes such as Na⁺/K⁺ pump activity in fibroblasts may reflect important events related to the initiation of DNA synthesis, it was observed that in both 3T3-4 and C3H-1 0T½ cells there were parallel increases in ³H-TdR incorporation and ouabain-sensitive ⁸⁶Rb⁺ influxes with 10^?7 M TPA, whereas PGF_2α stimulated a significant increase in ³H-TdR incorporation in 3T3-4 but not C3H-10T½ cells and only marginal increases in ouabain-sensitive ⁸⁶Rb⁺ influx in both. Therefore, although there appears to be a close correlation between Na⁺/K⁺ pump activation and subsequent S-phase entry following TPA stimulation, a similar correlation for PGF_2α cannot be confirmed.     

Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

The membrane localization of the plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.     

The effect of salty diets and gradual transfer to sea water on osmotic adaptation, gill Na+, K+-ATPase activation, and survival of brook charr, Salvelinus fontinalis, Mitchill

The intensity and duration of the period of osmotic disturbance during introduction of brook charr into sea water were decreased by introducing the fish according to a gradient of salinity over a period of 6 days. Survival in summer increased from 25 to 90% with the use of a salinity gradient. However, kinetics and levels of activation of the gill Na⁺, K⁺-ATPase were not affected by the mode used for introducing brook charr into sea water. Neither was its level of activity modified by the use of a salted diet when the fish were in fresh water. The addition of 8 and 12% of salt to the diet prevented the plasma electrolyte surge of concentrations during the first days in sea water. In very cold water, survival rate was also drastically improved by giving an 8% salted diet during the 6 weeks preceding the introduction into sea water. These results show that both salty diets and exposure to brackish water during 6 days help brook charr face osmotic stress and improve their survival rate when introduced into full-strength sea water. The combined use of these preconditioning strategies might facilitate rearing this species in sea cages or silos.     

Sodium-induced calcium deficiency in salt-stressed corn

Abstract The effect of the Na⁺/Ca²⁺ ratio in the root media on salt-stressed corn (Zea mays L. cvs DeKalb XL-75 and Pioneer 3906) was determined in greenhouse experiments. Plants grown in a complete nutrient solution salinized with 86.5 mol m^?3 NaCl exhibited severe Ca²⁺ deficiency symptoms at the four-leaf stage. The symptoms disappeared when part of the NaCl was replaced with 10 mol m^?3 CaCl₂ (Na⁺/Ca²⁺ molar ratio = 5.7). Salt stress at an iso-osmotic potential of ?0.4 MPa substantially decreased shoot growth at all solution Na⁺/Ca²⁺ ratios from 34.6 to 0.26. However, the dry weights of blades at 26 d of age were much less when plants were salinized with NaCl alone, particularly that of DeKalb XL-75 which was more susceptible to Na-induced Ca²⁺ deficiency than was Pioneer 3906. The growth of sheaths was similarity reduced by sail stress at all Na⁺/Ca²⁺ ratios. The symptoms of Ca²⁺ deficiency were correlated with low Ca²⁺ concentrations in the leaf tissue. Ca²⁺ concentrations in the developing blades of NaCl-stressed plants were much lower than in control plants. As the Na⁺/Ca²⁺ ratio in the solution was decreased, Ca²⁺ levels increased in both the blades and sheaths while Na⁺ concentrations greatly decreased. DeKalb XL-75 was much less effective than Pioneer 3906 in restricting the uptake of Na⁺. The results clearly indicate that NaCl stress may cause lesions and unique plant responses that are not manifested on agronomic plants grown on saline soils.     

Ion selectivity of epithelial Na channels

Epithelial Na channels are apparently pore-forming membrane proteins which conduct Na much better than any other biologically abundant ion. The conductance to Na can be 100 to 1000 times higher than that to K. The only other ions that can readily get through this channel are protons and Li. Small organic cations cannot pass through the channel, and water may also be impermeant. The selectivity properties of epithelial Na channels appear to be determined by at least three factors: A high field-strength anionic site, most likely a carboxyl residue of glutamic or aspartic acid residues on the channel protein, probably accounts for the high conductance through these channels of Na and Li and to the low conductance of K, Rb and Cs. A restriction in the size of the pore at its narrowest point probably accounts for the low conductance of organic cations as well as the possible exclusion of water molecules. The outer mouth of the channel appears to be negatively charged and may control access to the region of highest selectivity and may serve as a preliminary selectivity filter, attracting cations over anions. These conclusions are illustrated by the cartoon of the channel in Fig. 3. This picture is obviously both fanciful and simplified, but its general points will hopefully be testable. It leaves open a number of important questions, including: does amiloride block the channel by binding within the outer mouth? what does the inner mouth of the channel look like, and does this part of the channel contribute to selectivity? and what, if any, are the interactions between the features of the channel that impart selectivity and those that control the regulation of the channel by hormonal and other factors?     

Fast charge translocations associated with partial reactions of the Na,K-pump: II. Microscopic analysis of transient currents

Summary Nonstationary pump currents which have been observed in K⁺-free Na⁺ media after activation of the Na,K-ATPase by an ATP-concentration jump (see the preceding paper) are analyzed on the basis of microscopic reaction models. It is shown that the behavior of the current signal at short times is governed by electrically silent reactions preceding phosphorylation of the protein; accordingly, the main information on charge-translocating processes is contained in the declining phase of the pump current. The experimental results support the Albers-Post reaction scheme of the Na,K-pump, in which the translocation of Na⁺ precedes translocation of K⁺. The transient pump current is represented as the sum of contributions of the individual transitions in the reaction cycle. Each term in the sum is the product of a net transition rate times a dielectric coefficient describing the amount of charge translocated in a given reaction step. Charge translocation may result from the motion of ion-binding sites in the course of conformational changes, as well as from movement of ions in access channels connecting the binding sites to the aqueous media. A likely interpretation of the observed nonstationary currents consists in the assumption that the principal electrogenic step is the E₁-P/P-E₂ conformational transition of the protein, followed by a release of Na⁺ to the extracellular side. This conclusion is supported by kinetic data from the literature, as well as on the finding that chymotrypsin treatment which is known to block the E₁-P/P-E₂ transition abolishes the current transient. By numerical simulation of the Albers-Post reaction cycle, the proposed mechanism of charge translocation has been shown to reproduce the experimentally observed time behavior of pump currents.     

Distribution of calcium ATPase in the sarcoplasmic reticulum of fast- and slow-twitch muscles determined with monoclonal antibodies

Summary Four monoclonal antibodies against the calcium ATPase in sarcoplasmic reticulum (SR) of rabbit fast-twitch skeletal muscle were characterized using SDS-PAGE, Western blots and immunofluorescence. The ultrastructural distribution of the antigens was determined using post-embedding immunolabeling. The antibodies recognized the calcium ATPase in the SR but not in transverse (T-) tubule or plasma membranes. The antibody, D12, had the same binding affinity for the calcium ATPase from fast-twitch (rabbit sternomastoid) and slow-twitch (rabbit soleus) fibers and the affinity fell by 30% after fixation for electron microscopy in both types of muscle fiber. Ultrastructural studies revealed that the density of D12 antibody binding to the terminal cisternae membrane of extensor digitorum longus (edl) and sternomastoid fibers was on average seven times greater than in the slow-twitch soleus and semimembranosus fibers. Since the affinity of the ATPase for the antibody was the same in SR from fast- and slow-twitch muscles, the concentration of calcium ATPase in the terminal cisternae membrane of fast-twitch fibers was seven times greater than in slow-twitch fibers. This conclusion was supported by the fact that the concentration of calcium ATPase in light SR membranes was six times greater in SR from fast-twitch fibers than in SR from slow-twitch fibers. The results provide strong evidence that the different calcium accumulation rates in mammalian fast- and slow-twitch muscles are due to different concentrations of calcium ATPase molecules in the SR membrane.     

Effects of α2-Adrenergic Agonists and Antagonists on Photoreceptor Membrane Currents

Type B photoreceptors of the nudibranch mollusc Hermissenda crassicornis receive excitatory synaptic potentials (EPSPs) whose frequency is controlled by potential changes of a neighboring cell known as the S optic ganglion cell which is thought to be electrically coupled to the presynaptic source of these EPSPs, the E optic ganglion cell. The frequency of the EPSPs increases when a conditioned stimulus (light) is paired with an unconditioned stimulus (rotation) during acquisition of a Pavlovian conditioned response. The results of the present study are consistent with an adrenergic origin for these EPSPs. Noradrenergic agonists (greater than 100 microM), norepinephrine and clonidine, only slightly depolarize the type B cell but clearly prolong its depolarizing response to light. Serotonin, by contrast, causes hyperpolarization of the type B cell's resting potential as well as after a light step. Clonidine reduces voltage-dependent outward K+ currents (IA, an early current, ICa2+-K+, a late Ca2+-dependent current) that control the type B cell's excitability (and thus its light response and membrane potential). These effects of clonidine are reduced or blocked by the alpha 2-receptor antagonist, yohimbine (0.5 microM), but not the alpha 1-blocker, prazosin. The same yohimbine concentration also blocked depolarizing synaptic excitation of the type B cell in response to depolarization of a simultaneously impaled S optic ganglion cell. Histochemical techniques (both the glyoxylic acid method of de la Torre and Surgeon and the formaldehyde-induced fluorescence or Falck-Hillarp method) demonstrated the presence of a biogenic amine(s) within a single neuron in each optic ganglion as well as three or four cells within the vicinity of previously identified visual interneurons. No serotonergic neurons were found within the optic ganglion or in proximity to visual interneurons. A clonidine-like synaptic effect on type B cells, therefore, could amplify conditioning-specific changes of membrane currents by increasing type B depolarization and possibly, as well, by elevating intracellular second messengers.