Single chloride-permeable channels of large conductance in cultured cardiac cells of new-born rats

Large conductance channels were observed in the membrane of cultured cardiac cells of newborn rats studied with the patch-clamp technique in cell-attached and inside-out configurations. These channels were observed in 4% of the patches. In the cell-attached configuration they exhibited outward rectification and partial inactivation. In the inside-out configuration no rectification occurred but inactivation was present, mainly during hyperpolarizations. Two channels with large single unit conductances (400–450 pS) and one with a smaller conductance (200–250 pS) were frequently observed in the same patch. The two large channels generally had different kinetics. Under steady-state conditions the opening probability of the faster channel appeared to be voltage-independent. The slower channel was activated by depolarization. In asymmetrical solutions the permeability ratios P _Na/P _Cl were 0.03 and 0.24 for the larger and smaller channels, respectively; corresponding values for P _Ba/P _Cl were 0.04 and 0.09. It is proposed that in cardiac membranes the chloride permeability system is composed of widely dispersed microclusters forming grouped channels of different types and sizes.     

A physical model of sodium channel gating

Most current models of membrane ion channel gating are abstract compartmental models consisting of many undefined states connected by rate constants arbitrarily assigned to fit the known kinetics. In this paper is described a model with states that are defined in terms of physically plausible real systems which is capable of describing accurately most of the static and dynamic properties measured for the sodium channel of the squid axon. The model has two components. The Q-system consists of charges and dipoles that can move in response to an electric field applied across the membrane. It would contain and may compose the gating charge that is known to transfer prior to channel opening. The N-system consists of a charged group or dipole that is constrained to move only in the plane of the membrane and thus does not interact directly with the trans-membrane electric field but can interact electrostatically with the Q-system. The N-system has only two states, its resting state (channel closed) and its excited state (channel open) and its response time is very short in comparison with that of the Q-system. On depolarizing the membrane the the N-system will not make a transition to its open state until a critical amount of Q-charge transfer has occurred. Using only four adjustable parameters that are fully determined by fitting the equilibrium properties of the model to those of the sodium channel in the squid axon, the model is then able to describe with some accuracy the kinetics of channel opening and closing and includes the Cole and Moore delay. In addition to these predictions of the behaviour of assemblies of channels the model predicts some of the individual channel properties measured by patch clamp techniques.     

Activation of Dopamine Receptors Does Not Affect Phosphoinositide Turnover in NCB-20 Cells

Dopamine inhibits and serotonin stimulates adenylate cyclase activity in a neuroblastoma X Chinese hamster brain explant cell line (NCB-20). The inhibition of cyclic AMP accumulation by dopamine was blocked by pretreatment of the cells with pertussis toxin. Carbachol and bradykinin stimulated the accumulation of water-soluble inositol phosphates whereas thyrotropin-releasing hormone, vasopressin, neurotensin, and phenylephrine were without effect. Dopamine and serotonin had no significant effect on carbachol-induced phosphoinositide hydrolysis or the levels of the parent lipids within the membrane. Forskolin induced a much larger stimulation of cyclic AMP than did serotonin, and caused an increase in the levels of phosphatidylinositol-4-phosphate and phosphatidyl inositol-4,5-bisphosphate in the cell membrane.     

Characterization of the Actions of Toxins II-9.2.2 and II-10 from the Venom of the Scorpion Centruroides noxius on Transmitter Release from Mouse Brain Synaptosomes

Toxic peptides II-9.2.2 and II-10, purified from Centruroides noxius venom, bear highly homologous N-terminal amino acid sequences, and both toxins are lethal to mice. However, only toxin II-10 is active on the voltage-clamped squid axon, selectively decreasing the voltage-dependent Na+ current. Here, we have tested toxins II-9 and II-10 on synaptosomes from mouse brain: both toxins increased the release of gamma-[3H]aminobutyric acid ([3H]GABA). Their effect was completely blocked by tetrodotoxin or by the absence of external Na+. Also, both toxins increased Na+ permeability in isolated nerve terminals. Besides the observation that toxin II-9 is active on synaptosomes, the effect of toxin II-10 in this preparation is opposite to that observed in the squid axon. Thus, our results reflect functional differences between the populations of Na+ channels in mouse brain synaptosomes and in the squid axon. The release of GABA evoked by these toxins from synaptosomes required external Ca2+ and was blocked by Ca2+ channel blockers (verapamil and Co2+). This latter observation is in sharp contrast to the releasing action of veratrine, which evoked release even in the absence of external Ca2+. Furthermore, the action of both C. noxius toxins was potentiated by veratrine, a result suggesting they have different mechanisms of action. Among drugs that release neurotransmitters by increasing Na+ permeability, it is noteworthy that scorpion toxins are the only ones yet reported to have a strict requirement for external Ca2+.     

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

Ionic balance,proton efflux,nitrate reductase activity and growth ofHippophaë rhamnoides L. ssp.rhamnoides as influenced by combined-N nutrition or N2 fixation

Growth of 2-month-old nonnodulatedHippophaë rhamnoides seedlings supplied with combined N was compared with that of nodulated seedlings grown on zero N. Plant growth was significantly better with combined N than with N₂ fixation and, although not statistically significant for individual harvests, tended to be highest in the presence of NH ₄ ⁺ , a mixture of NH ₄ ⁺ and NO ₃ ^? producing the highest yields. Growth was severely reduced when solely dependent on N₂ fixation and, unlike the combined-N plants, shoot to root ratios had only slightly increased after an initial decrease. An apparently insufficient nodule mass (nodule weight ratio <5 per cent) during the greater part of the experimental period is suggested as the main cause of the growth reduction in N₂-fixing plants. Thein vivo nitrate reductase activity (NRA) of NO ₃ ^? dependent plants was almost entirely located in the roots. However, when grown with a combination of NO ₃ ^? and NH ₄ ⁺ , root NRA was decreased by approximately 85 per cent.H. rhamnoides demonstrated in the mixed supply a strong preference for uptake of N as NH ₄ ⁺ , NO ₃ ^? contributing only for approximately 20 per cent to the total N assimilation. Specific rates of N acquisition and ion uptake were generally highest in NO ₃ ^? +NH ₄ ⁺ plants. The generation of organic anions per unit total plant dry weight was approximately 40 per cent less in the NH ₄ ⁺ plants than in the NO ₃ ^? plants. Measured extrusions of H⁺ or OH^? (HCO ₃ ^? ) were generally in good agreement with calculated values on the basis of plant composition, and the acidity generated with N₂ fixation amounted to 0.45–0.55 meq H⁺. (mmol N_org)^?1. Without acidity control and in the presence of NH ₄ ⁺ , specific rates of ion uptake and carboxylate generation were strongly depressed and growth was reduced by 30–35 per cent. Growth of nonnodulatedH. rhamnoides plants ceased at the lower pH limit of 3.1–3.2 and deterioration set in; in the case of N₂-fixing plants the nutrient solution pH stabilized at a value of 3.8–3.9 without any apparent adverse effects upon plant performance. The chemical composition of experimental and field-growing plants is being compared and some comments are made on the nitrogen supply characteristics of their natural sites.     

Calcium-activated protease in the giant algaChara australis

Summary There is a protease, which is activated by Ca²⁺ (about 100 M), works at neutral pH and exists in the cytoplasm inChara australis. This protease may correspond to calpain, the calcium-activated neutral protease, which has been studied in animal cells. This is the first report showing the existence of a calcium-activated protease in plant cells.     

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?     

Demonstration and characterization of Ca2+ channel in tonoplast-free cells ofNitellopsis obtusa

Summary The presence of a Ca²⁺ channel in the plasmalemma of tonoplast-freeNitellopsis obtusa cells was demonstrated and its characteristics were studied using current- and voltage-clamp techniques. A long-lasting inward membrane current (I _m ), recorded using a step voltage clamp, consisted of a single component without time-dependent inactivation. Increasing either [Ca²⁺] _o or [Cl^–] _o 1) enhanced the maximum amplitude of inwardI _m ((I _m ) _p ) and 2) shifted the peak voltage ((V _m ) _p ) at(I _m ) _p to more positive values under ramp-shaped voltage clamping and 3) depolarized the peak value of action potentials. This behavior is consistent with predictions based on the Nernst equation for Ca²⁺ but not for Cl^–. DIDS (4,4-diisothiocyano-2,2-disulfonic acid stilbene) did not suppress(I _m ) _p in tonoplast-free cells, in contrast with its effect on normal cells. La³⁺ and nifedipine blocked(I _m ) _p irreversibly. On the other hand, Ca²⁺ channel agonist, BAY K 8644 irreversibly enhanced(I _m ) _p . Both Sr²⁺ influx and K⁺ efflux increased upon excitation. The charge carried by Sr²⁺ influx was compensated for by K⁺ efflux. It is concluded that only the Ca²⁺ channel is activated during plasmalemma excitation in tonoplast-free cells. In terms of the magnitude of(I _m ) _p , Sr²⁺ could replace Ca²⁺, but Mn²⁺, Mg²⁺ and Ba²⁺ could not. External pH affected(I _m ) _p and the membrane conductance (g _m ) at(I _m ) _p ((g _m ) _p ). Increasing the external ionic strength caused increases in both(I _m ) _p and(g _m ) _p , and shifted(V _m ) _p to positive values. At the same time, Sr²⁺ influx increased. Thus Ca²⁺ channel activation seems to be enhanced by increasing external ionic strength. The possible involvement of surface potential is discussed.     

Voltage gating in VDAC is markedly inhibited by micromolar quantities of aluminum

Summary The mitochondrial outer membrane contains voltagegated channels called VDAC that are responsible for the flux of metabolic substrates and metal ions across this membrane. The addition of micromolar quantities of aluminum chloride to phospholipid membranes containing VDAC channels greatly inhibits the voltage dependence of the channels' permeability. The channels remain in their high conducting (open) state even at high membrane potentials. An analysis of the change in the voltage-dependence parameters revealed that the steepness of the voltage dependence decreased while the voltage needed to close half the channels increased. The energy difference between the open and closed states in the absence of an applied potential did not change. Therefore, the results are consistent with aluminum neutralizing the voltage sensor of the channel. pH shift experiments showed that positively charged aluminum species in solution were not involved. The active form was identified as being either (or both) the aluminum hydroxide or the tetrahydroxoaluminate form. Both of these could reasonably be expected to neutralize a positively charged voltage sensor. Aluminum had no detectable effect of either single-channel conductance or selectivity, indicating that the sensor is probably not located in the channel proper and is distinct from the selectivity filter.