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.     

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+.     

Molecular cloning and physical characterization of a Brassica linear mitochondrial plasmid

Summary A linear mitochondrial plasmid reported to be associated with cytoplasmic male sterility in the genus Brassica was analyzed. A protein was found to be associated with the 5 ends of the plasmid. The entire plasmid was cloned by the homopolymer tailing technique via free hydroxyl groups present at its 3 ends. DNA sequence analysis of the cloned plasmid revealed a perfect terminal inverted repeat of 325 base pairs. Southern hybridization and restriction enzyme mapping analysis confirmed colinearity of the native plasmid and the clone, which showed significant homology with organelle DNA but not with nuclear DNA. Under high-stringency hybridization conditions, an internal 4.6 kb fragment of the 11.5 kb plasmid hybridized to the main mitochondrial genome in several species. Although the hybridization signal was weaker, the chloroplast genome also showed homology to the mitochondrial plasmid. The plasmid was undetectable at a molar ratio of less than 1/10 000 of the main mitochondrial genome in some lines of Brassica and Raphanus that contain the Ogura male sterile cytoplasm (cms). The absence of the plasmid in these sterile lines demonstrates that the plasmid is not required for the expression and maternal inheritance of male sterility.     

A comparative study of ammonium toxicity at different constatn pH of the nutrient solution

Seedlings of 14 species were grown for 14–28 days on nutrient solution with 6 mmol.l⁻¹ NH₄ as the sole nitrogen source. Solutions acidity was were kept constant at pH 4.0, 5.0, 6.0 and 7.0 by continuous titration with diluted KOH. The following species were used: barley, maize, oats sorghum, yellow and white lupin, pea, soybean, carrot, flax, castor-oil, spinach, sugarbeet and sunflower. Most plant species grew optimally at pH 6.0 with slight reductions at pH 5.0. Growth of many species was severely inhibited at pH 4.0, but this inhibition was not observed with the legume and cereal species. Yield depressions at pH 4.0 relative to pH 6.0 were well correlated with the respective relative decreases of the K concentration in their roots (P<0.002). In the roots of two species (sunflower and flax) total N concentrations were also strongly reduced at pH 4.0. apparently, the interactions between uptake of K, NH₄ and H ions become the prevalent problem at suboptimal pH. At pH 7.0, yields were also considerably decreased, with the exception of the lupines. At this pH, the roots of the growth inhibited plants were characterized by increased levels of total N and free NH₄. It is thought that the binding capacity of the roots for NH₄ is an important factor in preventing NH₄/NH₃ toxicity at supraoptimal pH.     

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.     

Modification of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells by N-bromoacetamide

Summary Ca²⁺-activated K⁺ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca²⁺ activation site was modified using N-bromoacetamide (NBA). 1mm NBA in the bath solution, at 2.5 m Ca²⁺ reduces the open probability,P _o , of the channel to <0.01, without an effect on single-channel conductance. NBA-modified channels are still Ca²⁺-sensitive, requiring 25mm Ca²⁺ to raiseP _o to 0.2. Both before and after NBA modification channel openings display at least two distributions, indicative of more than one open state. High Ca²⁺ (1mm) protects the channels from modification. Also presented is a second class of Ca²⁺-activated K⁺ channels which are normally present in MTAL cells which open infrequently at 10 m Ca²⁺ (P _o =0.01) but have aP _o of 0.08 at 1mm Ca²⁺. We can conclude (i) that NBA modifies the channel by shifting Ca²⁺-sensitivity to very high Ca²⁺, (ii) that NBA acts on a site involved in Ca²⁺ gating, and (iii) that a low affinity channel is present in the apical cell membrane with characteristics similar to those of normal channels modified with NBA.     

Effects of reagents modifying carboxyl groups on the gating current of the myelinated nerve fiber

Summary K⁺ channels in inside-out patches from hamster insulin tumor (HIT) cells were studied using the patch-clamp technique. HIT cells provide a convenient system for the study of ion channels and insulin secretion. They are easy to culture, form gigaohm seals readily and secrete insulin in response to glucose. The properties of the cells changed with the passage number. For cell passage numbers 48 to 56, five different K⁺-selective channels ranging from 15 to 211 pS in symmetrical 140mm KCl solutions were distinguished. The channels were characterized by the following features: a channel with a conductance (in symmetrical 140mm KCl solutions) of 210 pS that was activated by noncyclic purine nucleotides and closed by H⁺ ions (pH=6.8); a 211 pS channel that was Ca²⁺-activated and voltage dependent; a 185 pS channel that was blocked by TEA but was insensitive to quinine or nucleotides; a 130 pS channel that was activated by membrane hyperpolarization; and a small conductance (15 pS) channel that was not obviously affected by any manipulation. As determined by radioimmunoassay, cells from passage number 56 secreted 917±128 ng/mg cell protein/48 hr of insulin. In contrast, cells from passage number 77 revealed either no channel activity or an occasional nonselective channel, and secreted only 29.4±8.5 ng/mg cell protein/48 hr of insulin. The nonselective channel found in the passage 77 cells had a conductance of 25 pS in symmetrical 140mm KCl solutions. Thus, there appears to be a correlation between the presence of functional K⁺ channels and insulin secretion.     

Volume regulation in the early proximal tubule of theNecturus kidney

Summary The ability of early proximal tubule cells of theNecturus kidney to regulate volume was evaluated using light microscopy, video analysis and conventional microelectrodes.Necturus proximal tubule cells regulate volume in both hyperand hyposmotic solutions. Volume regulation in hyperosmotic fluids is HCO ₃ ^– dependent and is associated with a decrease in the relative K⁺ conductance of the basolateral cell membrane and a decrease in the resistance ratio,R _a /R _bl . Volume regulation in hyposmotic solutions is also dependent upon the presence of HCO ₃ ^– but is also inhibited by 2mm Ba²⁺ in the basolateral solution. Hyposmotic regulation is accompanied by an increase in the relative K⁺ conductance of the basolateral cell membrane and an increase inR _a /R _bl . Neither hypo- nor hyposmotic regulation have any affect on the depolarization of the basolateral cell membrane potential induced by HCO ₃ ^– removal. We conclude that volume regulation in the early proximal tubule of the kidney involves both HCO ₃ ^– -dependent transport systems and the basolateral K⁺ conductance.     

Solubilization and Characterization of the Nitrendipine Receptor in Rat Brain

The nitrendipine receptor associated with the voltage-dependent calcium channel in rat brain was solubilized by detergent extraction and sonication. The detergent solution used for extraction consisted of 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.25% (wt/vol) polyoxyethylene 20 cetyl ether (Brij 58), and 0.025% (wt/vol) polyoxyethylene 17 cetyl stearyl ether (Lubrol WX) in the presence of 30% (wt/vol) glycerol as a stabilizer. The molecular weight of the receptor was estimated to be 1,800K by Sephacryl S-500 gel filtration and 800K by sucrose density gradient sedimentation. The equilibrium dissociation constant of [3H]nitrendipine to the solubilized receptors was 5.6 nM, which is approximately 10 times that of the membrane-bound receptor. The binding of nitrendipine to the receptor was inhibited noncompetitively by the structurally unrelated calcium channel inhibitors verapamil and prenylamine; their concentrations for 50% inhibition were both 1.0 X 10(-7) M, and they caused maximal inhibitions of 70 and 100%, respectively.