Gating currents in the node of Ranvier: voltage and time dependence.

Like the axolemma of the giant nerve fibre of the squid, the nodal membrane of frog myelinated nerve fibres after blocking transmembrane ionic currents exhibits asymmetrical displacement currents during and after hyperpolarizing and depolarizing voltage clamp pulses of equal size. The steady-state distribution of charges as a function of membrane potential is consistent with Boltzmanns law (midpoint potential minus 33.7 mV; saturation value 17200 charges/mum-2). The time course of the asymmetry current and the voltage dependence of its time constant are consistent with the notion that due to a sudden change in membrane potential the charges undergo a first order transition between two configurations. Size and voltage dependence of the time constant are similar to those of the activation of the sodium conductance assuming m-2h kinetics. The results suggest that the presence of ten times more sodium channels (5000/mum-2) in the node of Ranvier than in the squid giant axon with similar sodium conductance per channel (2-3 pS).     

Extrasynaptic release of dopamine in a retinal neuron: activity dependence and transmitter modulation

Extrasynaptic release of dopamine is well documented, but its relation to the physiological activity of the neuron is unclear. Here we show that in absence of presynaptic active zones, solitary cell bodies of retinal dopaminergic neurons release by exocytosis packets of approximately 40,000 molecules of dopamine at irregular intervals and low frequency. The release is triggered by the action potentials that the neurons generate in a rhythmic fashion upon removal of all synaptic influences and therefore depends upon the electrical events at the neuronal surface. Furthermore, it is stimulated by kainate and abolished by GABA and quinpirole, an agonist at the D(2) dopamine receptor. Since the somatic receptors for these ligands are extrasynaptic, we suggest that the composition of the extracellular fluid directly modulates extrasynaptic release.     

Ionic dependence of amino-acid transport in the exocrine pancreatic epithelium: Calcium dependence of insulin action

Summary Rapid unidirectional transport (15 sec) ofl-serine and 2-methylaminoisobutyric acid (MeAIB) was studied in the isolated perfused rat pancreas using a dual-tracer dilution technique. Time-course experiments in the presence of normal cation gradients revealed a time-dependent transstimulation ofl-serine influx and transinhibition of MeAIB influx. Transport of the model nonmetabolized System A analog MeAIB was Na⁺ dependent and significantly inhibited during perfusion with 1mm ouabain. Although transport ofl-serine was largely Na⁺ independent, ouabain caused a time-dependent inhibition of transport. Influx of both amino acids appeared to be inhibited by the ionophore monensin but unaffected by a lowered extracellular potassium concentration. Removal of extracellular calcium had no effect on influx of the natural substratel-serine, whereas stimulation of transport by exogenous insulin (100 U/ml) was entirely dependent upon extracellular calcium and unaffected by ouabain. Paradoxically, exogenous insulin had no effect on the time-course of MeAIB influx. The characteristics ofl-serine influx described in earlier studies together with our present findings suggest that insulin may modulate the activity of System asc in the exocrine pancreatic epithelium by a calcium-dependent mechanism.     

Measurements of the BKCa channel's high-affinity Ca2+ binding constants: effects of membrane voltage

It has been established that the large conductance Ca²⁺-activated K⁺ channel contains two types of high-affinity Ca²⁺ binding sites, termed the Ca²⁺ bowl and the RCK1 site. The affinities of these sites, and how they change as the channel opens, is still a subject of some debate. Previous estimates of these affinities have relied on fitting a series of conductance–voltage relations determined over a series of Ca²⁺ concentrations with models of channel gating that include both voltage sensing and Ca²⁺ binding. This approach requires that some model of voltage sensing be chosen, and differences in the choice of voltage-sensing model may underlie the different estimates that have been produced. Here, to better determine these affinities we have measured Ca²⁺ dose–response curves of channel activity at constant voltage for the wild-type mSlo channel (minus its low-affinity Ca²⁺ binding site) and for channels that have had one or the other Ca²⁺ binding site disabled via mutation. To accurately determine these dose–response curves we have used a series of 22 Ca²⁺ concentrations, and we have used unitary current recordings, coupled with changes in channel expression level, to measure open probability over five orders of magnitude. Our results indicate that at −80 mV the Ca²⁺ bowl has higher affinity for Ca²⁺ than does the RCK1 site in both the opened and closed conformations of the channel, and that the binding of Ca²⁺ to the RCK1 site is voltage dependent, whereas at the Ca²⁺ bowl it is not.     

Adenosine: a neuron-glial transmitter promoting myelination in the CNS in response to action potentials

Neuronal activity influences myelination of the brain, but the molecular mechanisms involved are largely unknown. Here, we report that oligodendrocyte progenitor cells (OPCs) express functional adenosine receptors, which are activated in response to action potential firing. Adenosine acts as a potent neuron-glial transmitter to inhibit OPC proliferation, stimulate differentiation, and promote the formation of myelin. This neuron-glial signal provides a molecular mechanism for promoting oligodendrocyte development and myelination in response to impulse activity and may help resolve controversy on the opposite effects of impulse activity on myelination in the central and peripheral nervous systems.     

Nonequilibrium gating and voltage dependence of the ClC-0 Cl- channel

The gating of ClC-0, the voltage-dependent Cl- channel from Torpedo electric organ, is strongly influenced by Cl- ions in the external solution. Raising external Cl- over the range 1-600 mM favors the fast- gating open state and disfavors the slow-gating inactivated state. Analysis of purified single ClC-0 channels reconstituted into planar lipid bilayers was used to identify the role of Cl- ions in the channel's fast voltage-dependent gating process. External, but not internal, Cl- had a major effect on the channel's opening rate constant. The closing rate was more sensitive to internal Cl- than to external Cl-. Both opening and closing rates varied with voltage. A model was derived that postulates (a) that in the channel's closed state, Cl- is accessible to a site located at the outer end of the conduction pore, where it binds in a voltage-independent fashion, (b) that this closed conformation can open, whether liganded by Cl- or not, in a weakly voltage-dependent fashion, (c) that the Cl(-)-liganded closed channel undergoes a conformational change to a different closed state, such that concomitant with this change, Cl- ion moves inward, conferring voltage-dependence to this step, and (d) that this new Cl(-)- liganded closed state opens with a very high rate. According to this picture, Cl- movement within the pre-open channel is the major source of voltage dependence, and charge movement intrinsic to the channel protein contributes very little to voltage-dependent gating of ClC-0. Moreover, since the Cl- activation site is probably located in the ion conduction pathway, the fast gating of ClC-0 is necessarily coupled to ion conduction, a nonequilibrium process.     

Slo3 K+ channels: voltage and pH dependence of macroscopic currents

The mouse Slo3 gene (KCNMA3) encodes a K(+) channel that is regulated by changes in cytosolic pH. Like Slo1 subunits responsible for the Ca(2+) and voltage-activated BK-type channel, the Slo3 alpha subunit contains a pore module with homology to voltage-gated K(+) channels and also an extensive cytosolic C terminus thought to be responsible for ligand dependence. For the Slo3 K(+) channel, increases in cytosolic pH promote channel activation, but very little is known about many fundamental properties of Slo3 currents. Here we define the dependence of macroscopic conductance on voltage and pH and, in particular, examine Slo3 conductance activated at negative potentials. Using this information, the ability of a Horrigan-Aldrich-type of general allosteric model to account for Slo3 gating is examined. Finally, the pH and voltage dependence of Slo3 activation and deactivation kinetics is reported. The results indicate that Slo3 differs from Slo1 in several important ways. The limiting conductance activated at the most positive potentials exhibits a pH-dependent maximum, suggesting differences in the limiting open probability at different pH. Furthermore, over a 600 mV range of voltages (-300 to +300 mV), Slo3 conductance shifts only about two to three orders of magnitude, and the limiting conductance at negative potentials is relatively voltage independent compared to Slo1. Within the context of the Horrigan-Aldrich model, these results indicate that the intrinsic voltage dependence (z(L)) of the Slo3 closed-open equilibrium and the coupling (D) between voltage sensor movement are less than in Slo1. The kinetic behavior of Slo3 currents also differs markedly from Slo1. Both activation and deactivation are best described by two exponential components, both of which are only weakly voltage dependent. Qualitatively, the properties of the two kinetic components in the activation time course suggest that increases in pH increase the fraction of more rapidly opening channels.     

Regulation of voltage dependence of the KAT1 channel by intracellular factors

The KAT1 channel is a hyperpolarization-activated K+ channel cloned from the higher plant Arabidopsis. The deduced amino acid sequence suggests that its structural organization is similar to that of the Shaker-like K+ channel activated by depolarization. Electrophysiological properties of the KAT1 channel expressed in Xenopus oocytes indicate that voltage-dependent activation of the KAT1 channel is not caused by the divalent ion block and that it is intrinsic to the channel. Activity of the KAT1 channel progressively decreases upon patch excision. This rundown of the channel is accompanied by a large shift in the voltage dependence of the channel to a more negative direction. The voltage dependence is also regulated by pH, ATP, and cGMP.     

Methodological aspects of the problem of nonmonotonic dose-effect dependence

Considered are the methodological bases of two radiobiological theories—target theory and structural-metabolic theory. The target theory states the existence in the cell of a biologically significant structure (sensitive volume) and the monotonic “dose-effect“ dependence. In the structural-metabolic theory at the molecular level the unity of structure and metabolism is postulated. Discussed is the role of homeostatic mechanisms in the nonmonotonicity of the “time-effect” dependence. The nonmonotonicity of the “dose-effect” dependence for metabolic and functional parameters of cells and tissues is stipulated by the non-monotonicity in time of the metabolic and functional response to the action of ionizing radiation.     

Molecular action of lidocaine on the voltage sensors of sodium channels

Block of sodium ionic current by lidocaine is associated with alteration of the gating charge-voltage (Q-V) relationship characterized by a 38% reduction in maximal gating charge (Q(max)) and by the appearance of additional gating charge at negative test potentials. We investigated the molecular basis of the lidocaine-induced reduction in cardiac Na channel-gating charge by sequentially neutralizing basic residues in each of the voltage sensors (S4 segments) in the four domains of the human heart Na channel (hH1a). By determining the relative reduction in the Q(max) of each mutant channel modified by lidocaine we identified those S4 segments that contributed to a reduction in gating charge. No interaction of lidocaine was found with the voltage sensors in domains I or II. The largest inhibition of charge movement was found for the S4 of domain III consistent with lidocaine completely inhibiting its movement. Protection experiments with intracellular MTSET (a charged sulfhydryl reagent) in a Na channel with the fourth outermost arginine in the S4 of domain III mutated to a cysteine demonstrated that lidocaine stabilized the S4 in domain III in a depolarized configuration. Lidocaine also partially inhibited movement of the S4 in domain IV, but lidocaine's most dramatic effect was to alter the voltage-dependent charge movement of the S4 in domain IV such that it accounted for the appearance of additional gating charge at potentials near -100 mV. These findings suggest that lidocaine's actions on Na channel gating charge result from allosteric coupling of the binding site(s) of lidocaine to the voltage sensors formed by the S4 segments in domains III and IV.     

Hysteresis in the voltage dependence of HCN channels: conversion between two modes affects pacemaker properties

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are important for rhythmic activity in the brain and in the heart. In this study, using ionic and gating current measurements, we show that cloned spHCN channels undergo a hysteresis in their voltage dependence during normal gating. For example, both the gating charge versus voltage curve, Q(V), and the conductance versus voltage curve, G(V), are shifted by about +60 mV when measured from a hyperpolarized holding potential compared with a depolarized holding potential. In addition, the kinetics of the tail current and the activation current change in parallel to the voltage shifts of the Q(V) and G(V) curves. Mammalian HCN1 channels display similar effects in their ionic currents, suggesting that the mammalian HCN channels also undergo voltage hysteresis. We propose a model in which HCN channels transit between two modes. The voltage dependence in the two modes is shifted relative to each other, and the occupancy of the two modes depends on the previous activation of the channel. The shifts in the voltage dependence are fast (tau approximately 100 ms) and are not accompanied by any apparent inactivation. In HCN1 channels, the shift in voltage dependence is slower in a 100 mM K extracellular solution compared with a 1 mM K solution. Based on these findings, we suggest that molecular conformations similar to slow (C-type) inactivation of K channels underlie voltage hysteresis in HCN channels. The voltage hysteresis results in HCN channels displaying different voltage dependences during different phases in the pacemaker cycle. Computer simulations suggest that voltage hysteresis in HCN channels decreases the risk of arrhythmia in pacemaker cells.     

Inactivation of N-type calcium current in chick sensory neurons: calcium and voltage dependence

We have studied the inactivation of high-voltage-activated (HVA), omega- conotoxin-sensitive, N-type Ca2+ current in embryonic chick dorsal root ganglion (DRG) neurons. Voltage steps from -80 to 0 mV produced inward Ca2+ currents that inactivated in a biphasic manner and were fit well with the sum of two exponentials (with time constants of approximately 100 ms and > 1 s). As reported previously, upon depolarization of the holding potential to -40 mV, N current amplitude was significantly reduced and the rapid phase of inactivation all but eliminated (Nowycky, M. C., A. P. Fox, and R. W. Tsien. 1985. Nature. 316:440-443; Fox, A. P., M. C. Nowycky, and R. W. Tsien. 1987a. Journal of Physiology. 394:149-172; Swandulla, D., and C. M. Armstrong. 1988. Journal of General Physiology. 92:197-218; Plummer, M. R., D. E. Logothetis, and P. Hess. 1989. Neuron. 2:1453-1463; Regan, L. J., D. W. Sah, and B. P. Bean. 1991. Neuron. 6:269-280; Cox, D. H., and K. Dunlap. 1992. Journal of Neuroscience. 12:906-914). Such kinetic properties might be explained by a model in which N channels inactivate by both fast and slow voltage-dependent processes. Alternatively, kinetic models of Ca-dependent inactivation suggest that the biphasic kinetics and holding-potential-dependence of N current inactivation could be due to a combination of Ca-dependent and slow voltage- dependent inactivation mechanisms. To distinguish between these possibilities we have performed several experiments to test for the presence of Ca-dependent inactivation. Three lines of evidence suggest that N channels inactivate in a Ca-dependent manner. (a) The total extent of inactivation increased 50%, and the ratio of rapid to slow inactivation increased approximately twofold when the concentration of the Ca2+ buffer, EGTA, in the patch pipette was reduced from 10 to 0.1 mM. (b) With low intracellular EGTA concentrations (0.1 mM), the ratio of rapid to slow inactivation was additionally increased when the extracellular Ca2+ concentration was raised from 0.5 to 5 mM. (c) Substituting Na+ for Ca2+ as the permeant ion eliminated the rapid phase of inactivation. Other results do not support the notion of current-dependent inactivation, however. Although high intracellular EGTA (10 mM) or BAPTA (5 mM) concentrations suppressed the rapid phase inactivation, they did not eliminate it. Increasing the extracellular Ca2+ from 0.5 to 5 mM had little effect on this residual fast inactivation, indicating that it is not appreciably sensitive to Ca2+ influx under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

A soluble mitochondrial protein increases the voltage dependence of the mitochondrial channel,VDAC

A soluble protein isolated from mitochondria has been found to modulate the voltage-dependent properties of the mitochondrial outer membrane channel, VDAC. This protein, called the VDAC modulator, was first found inNeurospora crassa and then discovered in species from other eukaryotic kingdoms. The modulator-containing fraction (at a crude protein concentration of 20 µg/ml) increases the voltage dependence of VDAC channels over 2–3-fold. At higher protein concentrations (50–100 µg/ml), some channels seem to remain in a closed state or be blocked while others display the higher voltage dependence and are able to close at low membrane potentials. By increasing the steepness of the voltage-dependent properties of VDAC channels, this modulator may serve as an amplifierin vivo to increase the sensitivity of the channels in response to changes in the cell's microenvironment, and consequently, regulate the metabolic flux across the outer mitochondrial membrane by controlling the gating of VDAC channels.