The nature of the voltage-dependent conductance of the hemocyanin channel.

The electrical responses of individual hemocyanain channels in oxidized cholesterol membranes demonstrate that the voltage-dependent conductance of many-chanel membranes arises from two different mechanisms. These are the voltage-dependent redistribution of channels among several discrete single-channel conductance states themselves. The relaxation time for the discrete conductance changes is of the order of seconds nd the relaxation time of the continuous conductance changes is of the order 10(-4) seconds. As salt concentration in the bathing medium is increased, the single-channel conductance first increases lineary and then saturates. The characteristics of the saturation curves suggest that the continuous conductance changes occur at the edges of the channel and that the mean time an ion spends in the channel is 4 nanoseconds...     

The nature of the voltage-dependent conductance induced by alamethicin in black lipid membranes

Summary Alamethicin induces a conductance in black lipid films which increases exponentially with voltage. At low conductance the increase occurs in discrete steps which form a pattern of five levels, the second and third being most likely. The conductance of each level is directly proportional to salt concentration, inversely proportional to solution viscosity, and nearly independent of voltage.The probability distribution of the five steps is not a function of voltage, but as the voltage is increased, more levels begin to appear. These can be explained as super-positions of the original five, both in position and relative probability.This suggests that the five levels are associated with a physical entity which we call a pore. This point of view is confirmed by the following measurements. The kinetic response of the current to a voltage step is first order, and shows an exponential increase in rate of pore formation and an exponential decrease in rate of pore disappearance with voltage. If these rates are statistical, the number of pores should fluctuate about a voltage-dependent mean. High conductance current fluctuations are too large to be explained by fluctuation in the number of pores alone. But if fluctuations among the five levels are included, the magnitude of the fluctuations at high conductance is accurately predicted.Alamethicin adsorbs reversibly to the membrane surface, and the conductance at a fixed voltage depends on the ninth power of alamethicin concentration and on the fourth power of salt concentration, in the aqueous phase. In our bacterial phosphatidyl ethanolamine membranes, alamethicin added to one side of the membrane produces elevated conductance only when the voltage on that side is increased.On leave of absence from the Facultad de Ciencias, Universidad de Chile, Santiago de Chile.     

The voltage-dependent anion channel

Recently, it has been recognized that there is a metabolic coupling between the cytosol and mitochondria, where the outer mitochondrial membrane (OMM), the boundary between these compartments, has important functions. In this crosstalk, mitochondrial Ca2+ homeostasis and ATP production and supply play a major role. The primary transporter of ions and metabolites across the OMM is the voltage-dependent anion channel (VDAC). The interaction of VDAC with Ca2+, ATP glutamate, NADH, and different proteins was demonstrated, and these interactions may regulate OMM permeability. This review includes information on VDAC purification methods, characterization of its channel activity (selectivity, voltage-dependence, conductance), and the regulation of VDAC channel by ligands, such as Ca2+, glutamate and ATP and touches on many aspects of the physiological relevance of VDAC to Ca2+ homeostasis and mitochondria-mediated apoptosis.     

The dependence of the conductance of the hemocyanin channel on applied potential and ionic concentration with mono- and divalent cations

Incorporation of Megatura crenulata hemocyanin into phosphatidylcholine black lipid membranes results in the formation of ion channels. Channel properties depend on many factors, three of which are examined in this work: type and concentration of electrolyte and applied voltage. Eight cations at different concentrations have been used. Instantaneous conductance of the channel is a saturating function of both applied voltage and ionic strength of the bathing solution with monovalent cations, but only of ionic strength with divalent cations. Steady-state voltage-conductance relations are nonlinear for both signs but show slight saturation with ionic strength. Relaxation towards the steady state can be fitted by two exponentials with different time constants. All experimental data are fitted postulating the existence of a mechanism of voltage gating of the channel, and of discrete negative charge near its mouth. Specific and nonspecific binding of cations is required.     

Multiple conductance channel activity of wild-type and voltage-dependent anion-selective channel (VDAC)-less yeast mitochondria.

Yeast mitoplasts (mitochondria with the outer membrane stripped away) exhibit multiple conductance channel activity (MCC) in patch-clamp experiments that is very similar to the activity previously described in mammalian mitoplasts. The possible involvement of the voltage-dependent anion-selective channel (VDAC) of the outer membrane in MCC activity was explored by comparing the channel activity in wild-type yeast mitoplasts with that of a VDAC-deletion mutant. The channel activity recorded from the mutant is essentially the same as that of the wild-type in the voltage range of -40 to 30 mV. These observations indicate that VDAC is not required for MCC activity. Interestingly, the channel activity of the VDAC-less yeast mitoplasts exhibits altered gating properties at transmembrane potentials above and below this range. We conclude that the deletion of VDAC somehow results in a modification of MCC's voltage dependence. In fact, the voltage profile recorded from the VDAC-less mutant resembles that of VDAC.     

The intrinsically disordered N-terminus of the voltage-dependent anion channel

The voltage-dependent anion channel (VDAC) is a critical β-barrel membrane protein of the mitochondrial outer membrane, which regulates the transport of ions and ATP between mitochondria and the cytoplasm. In addition, VDAC plays a central role in the control of apoptosis and is therefore of great interest in both cancer and neurodegenerative diseases. Although not fully understood, it is presumed that the gating mechanism of VDAC is governed by its N-terminal region which, in the open state of the channel, exhibits an α-helical structure positioned midway inside the pore and strongly interacting with the β-barrel wall. In the present work, we performed molecular simulations with a recently developed force field for disordered systems to shed new light on known experimental results, showing that the N-terminus of VDAC is an intrinsically disordered region (IDR). First, simulation of the N-terminal segment as a free peptide highlighted its disordered nature and the importance of using an IDR-specific force field to properly sample its conformational landscape. Secondly, accelerated dynamics simulation of a double cysteine VDAC mutant under applied voltage revealed metastable low conducting states of the channel representative of closed states observed experimentally. Related structures were characterized by partial unfolding and rearrangement of the N-terminal tail, that led to steric hindrance of the pore. Our results indicate that the disordered properties of the N-terminus are crucial to properly account for the gating mechanism of VDAC.     

Kinetic properties of a voltage-dependent junctional conductance

We have proposed that the gap junctions between amphibian blastomeres are comprised of voltage-sensitive channels. The kinetic properties of the junctional conductance are here studied under voltage clamp. When the transjunctional voltage is stepped to a new voltage of the same polarity, the junctional conductance changes as a single exponential to a steady-state level. The time constant of the conductance change is determined by the existing transjunctional voltage and is independent of the previous voltage. For each voltage polarity, the relations between voltage, time constant, and steady-state conductance are well modeled by a reversible two-state reaction scheme in which the calculated rate constants for the transitions between the states are exponential functions of voltage. The calculated rate constant for the transition to the low-conductance state is approximately twice as voltage dependent as that for the transition to the high-conductance state. When the transjunctional voltage polarity is reversed, the junctional conductance undergoes a transient recovery. The polarity reversal data are well modeled by a reaction scheme in which the junctional channel has two gates, each with opposite voltage sensitivity, and in which an open gate may close only if the gate in series with it is open. A simple explanation for this contingent gating is a mechanism in which each gate senses only the local voltage drop within the channel.     

Equilibrium properties of a voltage-dependent junctional conductance

The conductance of junctions between amphibian blastomeres is strongly voltage dependent. Isolated pairs of blastomeres from embryos of Ambystoma mexicanum, Xenopus laevis, and Rana pipiens were voltage clamped, and junctional current was measured during transjunctional voltage steps. The steady-state junctional conductance decreases as a steep function of transjunctional voltage of either polarity. A voltage-insensitive conductance less than 5% of the maximum remains at large transjunctional voltages. Equal transjunctional voltages of opposite polarities produce equal conductance changes. The conductance is half maximal at a transjunctional voltage of approximately 15 mV. The junctional conductance is insensitive to the potential between the inside and outside of the cells. The changes in steady-state junctional conductance may be accurately modeled for voltages of each polarity as arising from a reversible two-state system in which voltage linearly affects the energy difference between states. The voltage sensitivity can be accounted for by the movement of about six electron charges through the transjunctional voltage. The changes in junctional conductance are not consistent with a current-controlled or ionic accumulation mechanism. We propose that the intramembrane particles that comprise gap junctions in early amphibian embryos are voltage-sensitive channels.     

Alkali metal ion selectivity of the hemocyanin channel

Summary The selectivity of the hemocyanin channel was measured for alkali metal ions and ammonium. Permeability ratios relative to K⁺ measured from biionic potentials were: NH ₄ ⁺ (1.52)>Rb⁺ (1.05)>K⁺ (1.0)>Cs⁺ (0.89)>Na⁺ (0.81)>Li⁺ (0.35). Single-channel ion conductance was a saturating function of ion concentration regardless of the cation present in the bathing medium. Maximal conductances were 270, 267, 215, 176, 170 and 37 ps for K⁺, Rb⁺, NH ₄ ⁺ , Cs⁺, Na⁺ and Li⁺, respectively. Current-voltage curves for the different monovalent cations were measured and described using a threebarrier model previously used to explain the voltage dependence of the instantaneous channel conductance (Cecchi, Alvarez & Latorre, 1981). In this way, binding and peak energies were estimated for the different ions. Considering the energy peaks as transition states between the ion and the channel, it is concluded that they follow Eisenman's selectivity sequences XI (cis peak, i.e., Li⁺>Na⁺>K⁺>Rb⁺>Cs⁺; highest field strength), VII (central peak) and II (trans peak). The cis side was that to which hemocyanin was added and was electrically ground. The binding energies, on the other hand, follow Eisenman's series XI for strong electric field sites. Binding of NH ₄ ⁺ to the cis-well suggests that the orientation of the ligands in the site is tetrahedric.     

A three-barrier model for the hemocyanin channel

Keyhole limpet hemocyanin forms ion-conducting channels in planar lipid bilayer membranes. Ionic current through the open hemocyanin channel presents the following characteristics: (a) it is carried mainly by cations; (b) it is a nonlinear function of membrane potential; (c) channel conductance is a saturating function of ion activity; (d) it shows ionic competition. A model for the open hemocyanin channel is developed from absolute reaction rate theory. The model calls for three energy barriers in the channel. Two energy barriers represent the entrance and exit of the ion into and out of the channel. The third barrier separates two energy minima that represent two binding sites. Furthermore, only one ion is allowed inside the channel at a given time. This model is able to recreate all the hemocyanin characteristics found experimentally in negatively charged and neutral membranes.     

Coupling of voltage-dependent gating and Ba++ block in the high- conductance, Ca++-activated K+ channel

Voltage-dependent Ca++-activated K+ channels from rat skeletal muscle were reconstituted into planar lipid bilayers, and the kinetics of block of single channels by Ba++ were studied. The Ba++ association rate varies linearly with the probability of the channel being open, while the dissociation rate follows a rectangular hyperbolic relationship with open-state probability. Ba ions can be occluded within the channel by closing the channel with a strongly hyperpolarizing voltage applied during a Ba++-blocked interval. Occluded Ba ions cannot dissociate from the blocking site until after the channel opens. The ability of the closed channel to occlude Ba++ is used as an assay to study the channel's gating equilibrium in the blocked state. The blocked channel opens and closes in a voltage-dependent process similar to that of the unblocked channel. The presence of a Ba ion destabilizes the closed state of the blocked channel, however, by 1.5 kcal/mol. The results confirm that Ba ions block this channel by binding in the K+-conduction pathway. They further show that the blocking site is inaccessible to Ba++ from both the cytoplasmic and external solutions when the channel is closed.     

The low conductance K(+) channel in human colonic crypt cells has a voltage-dependent permeability not affected by Mg(++)

The low conductance K(+) channel found in human colonocytes was investigated using the patch-clamp technique. The channel is Ca(++)-dependent and is blocked by Ba(++) (5 mM) with a decrease in open probability from 0.42 to 0.19. At -40 mV the slope conductance was 29 pS (using intracellular solution in the pipette). In inside-out patches, inward rectification was seen both with KCl (pipette)/NaCl (bath) solutions as well as KCl/KCl solutions. The rectification could not be affected by omitting Mg(++) from the pipette or the bath solution, neither by exposing the patches to the polyamine spermine (1 mM). Using the Goldman-Hodgkin-Katz equation we show that the permeability decreased in a linear fashion from approximately 5.2 x 10(-14) cm(3)/s to 1.8 x 10(-14) cm(3)/s (-100 to +100 mV), both with and without Mg(++) in the solutions. There was no significant difference in the nominal values of permeability. This property of the K(+) channel may facilitate the hyperpolarization needed to sustain a chloride secretion.     

Melittin induced voltage-dependent conductance in DOPC lipid bilayers

Melittin-induced conductance was measured on planar bilayers made from dioleoylphosphatidylcholine. Upon application of a fixed voltage, the current response was monophasic and remained so even after prolonged observation times. The conductance of melittin-doped bilayers increased exponentially with voltage. In addition, an ohmic contribution appeared after some current had passed. The voltage-dependent conductance increased e-fold every 22 mV and was proportional to the fourth power of the aqueous monomeric peptide concentration, for all salt concentrations investigated (0.4-1.8 M NaCl). Discrete conductance steps could be resolved at all these salt concentrations. The amplitudes of these steps were highly variable. In each experiment, conductance was initially only observed for potentials which were positive on the side of peptide addition. As more and more current passed across the bilayer, the current-voltage curves became symmetric. The system needed some time to reach stationary current-voltage characteristics: about 50 min at pH 7 but only about 15 min at pH 8, suggesting involvement of the N-terminus (pK around 7.5) of melittin in the slow formation of a 'prepore'.     

Voltage-dependent conductance induced by hemocyanin in black lipid films

When hemocyanin is added to a black lipid film, the conductance increases in discrete steps. For negative potentials the single step conductance is constant, but for positive potentials the step conductance appears to decrease as the potential increases. At high positive potentials the conductance fluctuates between several levels. These data suggest that, in lipid membranes, hemocyanin conducts ions through discrete channels. The voltage-dependent conductance observed at high levels of conductance seems to be a consequence of the properties of the conductance of the single channel.