Conductivity and diameter of latrotoxin channels in lipid bilayers

It is shown that approximately 9 A Ca-selective ion channels were induced in bilayer lipid membrane (BLM) from phosphatidylserine by nonpurified spider venom (Latrodectus tredicimguttatus) and by alpha-latrotoxin obtained from it. It is established that channels greatly different in conductivity have the same diameter and nearly the same charge constitution, that evidences for their claster organization. The purification process as well as freezing-thawing and long time keeping influence the channel conductivity without changing its diameter and selectivity.     

Immunochemical and functional study of the latrotoxin molecule]

A panel of monoclonal antibodies (mAb) against alpha-latrotoxin (LT) has been produced and their main characteristics have been determined. The influence of mAb on the functional effects of LT in synaptosomes from rat brain and on the channel formation in bilayer lipid membrane has been investigated. These mAbs do not inhibit binding of LT to rat synaptosomes but modify LT-receptor interaction in terms of LT's channel-forming and secretogenic effects. Antibodies A6 and A24 block these effects, whereas A4 partially preserves the secretogenic action of LT and completely blocks its channel-forming action. Only antibodies A15 affect the LT ability to form cationic channels in BLM, inducing considerable decrease in the frequency of the channel formation. These data and their analysis allow to identify several functional (and, probably, structural) domains of LT responsible for: 1) toxin-receptor interaction; 2) channel-forming and related calcium-dependent secretogenic effects; 3) calcium-independent secretogenic effects; 4) formation of cationic channels in the artificial lipid bilayer.     

Blocking of the beta-latrotoxin channels in bimolecular lipid membranes by antibodies

Asymmetric ion channels are formed in a bimolecular lipid membrane by beta-latrotoxin (LT) introduced to one (cis) side of the membrane. LT-specific antibodies added to the opposite (trans) side of the membrane block the current through the LT channels when a negative potential is applied to the cis side, no blockade is observed at positive potentials. LT-specific antibodies do not block the channel current when added to the cis compartment after removal of LT. LT-unspecific immunoglobulins have no influence on LT channel conductance.     

Voltage clamp analysis of two inward current mechanisms in the egg cell membrane of a starfish

Ionic mechanisms of excitation were studied in the immature egg cell membrane of a starfish, Mediaster aequalis, by analyzing membrane currents during voltage clamp. The cell membrane shows two different inward current mechanisms. One is activated at a membrane potential of -55 approximately -50 mV and the other at -7 approximately -6 mV. They are referred to as channels I and II, respectively. A similar difference is also found in the membrane potential of half inactivation. Currents of the two channels can, therefore, be separated by selective inactivation. The currents of both channels depend on Ca++ (Sr++ or Ba++) but only the current of channel I depends on Na+. The time-course of current differs significantly between the two channels when compared at the same membrane potential. The relationship between the membrane current and the concentration of the permeant ions is also different between the two channels. The result suggests that channel II is a more saturable system. The sensitivity of the current to blocking cations such as Co++ or Mg++ is substantially greater in channel II than in channel I. Currents of both channels depend on the external pH with an apparent pK of 5.6. They are insensitive to 3 muM tetrodotoxin (TTX) but are eliminated totally by 7.3 mM procaine. The properties of channel II are similar to those of the Ca channel found in various adult tissues. The properties of channel I differ, however, from those of either the typical Ca or Na channels. Although the current of the channel depends on the external Na the amplitude of the Na current decreases not only with the Na concentration but also with the Ca concentration. No selectivity is found among Li+, Na+, Rb+, and Cs+. The experimental result suggests that Na+ does not carry current but modifies the current carried by Ca in channel I.     

Study of channel-forming properties of latrotoxin in liposomes

Interaction of latrotoxin with phospholipid vesicles and bilayer lipid membranes is shown to proceed differently. Latrotoxin when interacting with liposomes is sorbed on the membrane surface forming no ionic channels in this case. Only latrotoxin fragments obtained due to the toxin hydrolysis by pronase or trypsin are able to form channels. These fragments being inserted into liposomes are coupled strongly with the membrane and are not subjected to the further splitting by proteinases. The electrophoretic spectrum of peptides bound with liposomes is presented by protein components with a molecular weight of 116, 100, 92, 67, 52 and 45 kDa, while zone typical of latrotoxin is absent in this spectrum. The method of small-angle X-rays scattering has shown that tryptic fragments of latrotoxin penetrate into the phospholipid bilayer of liposomes.     

Resonance phenomena in membranes containing ion channels with inactivation

A mathematical model of the ion transport across a membrane containing channel with inactivation has been analysed. Under certain conditions, such a membrane has been shown to behave as a selfoscillating circuit of a very high quality, its own frequency ranging for a variety of natural channels between 10(-1)-10(3) cycles. When exposed to an alternating electric field with a frequency approximating f0, the membrane displays resonance changes in its potential and channel conductivity. The average (over a period of forced oscillations) values of the potential and conductivity also show a resonance type of dependence on the frequency of the external field.     

Effect of calcium ions on potassium conductivity of latrotoxin channels

It has been shown that inhibition of potassium current through latrotoxin channels by calcium ions is followed by electrostatic interaction of these ions with a total charge on the mouth of the channel.     

Ion channels in rabbit cultured fibroblasts

Large outward currents are recorded with the whole-cell patch-clamp technique on depolarization of rabbit cultured fibroblasts. Our findings suggest that these outward currents consist of two voltage-dependent components, one of which also depends on cytoplasmic calcium concentration. Total replacement of external Cl- by the large anion ascorbate does not affect the amplitude of the currents, indicating that both components must be carried by K+. Consistent with these findings with whole-cell currents, in single channel recordings from fibroblasts we found that most patches contain high-conductance potassium-selective channels whose activation depends on both membrane potential and the calcium concentration at the cytoplasmic surface of the membrane. In a smaller number of patches, a second population of high-conductance calcium-independent potassium channels is observed having different voltage-dependence. The calcium- and voltage-dependence suggest that these two channels correspond with the two components of outward current seen in the whole-cell recordings. The single channel conductance of both channels in symmetrical KCl (150 mM) is 260-270 pS. Both channels are highly selective for K+ over both Na+ and Cl-. The conductance of the channels when outward current is carried by Rb+ is considerably smaller than when it is carried by K+. Some evidence is adduced to support the hypothesis that these potassium channel populations may be involved in the control of cell proliferation.     

A simple method for the determination of the pore radius of ion channels in planar lipid bilayer membranes

Abstract A new method of pore size determination is presented. The results of applying this simple method to ion channels formed by staphylococcal α-toxin and its N-terminal fragment as well as to cholera toxin channels are shown. The advantages and the difficulties of this method are discussed. It was found that (i) the mobility of ions in solutions depends only on the percentage of concentration of added non-electrolytes and practically not on their chemical nature (sugars or polyglycols) and molecular size; (ii) the proportional change of both ion channel conductance and bulk solution conductivity by low M . non-electrolytes may be used as an indication of a diffusion mechanism of ion transport through channels; (iii) the slope of the dependence of the ion channel conductance on the bulk conductivity of solutions containing different concentrations of non-electrolyte is a good measure of channel permeability for non-electrolytes.     

Properties of the channels induced by Lityphantes paykullianus venom

It was found that common venom of Lityphantes paykullianus is capable of forming conductivity channels on bilayer phospholipid membrane. The values of channel conductivity are remarkably distinguished in the solutions of different alkaline metals. The selectivity of channels is mainly cationic at pH 7.5 with weak discrimination of ions few tell one from the other by size. These results indicate that there is an active component in the common venom accountable for channels formation.     

Comparative analysis of cholesterol sensitivity of Kir channels: Role of the CD loop

Kir channels are important in setting the resting membrane potential and modulating membrane excitability. A common feature of Kir2 channels and several other ion channels that has emerged in recent years is that they are regulated by cholesterol, a major lipid component of the plasma membrane whose excess is associated with multiple pathological conditions. Yet, the mechanism by which cholesterol affects channel function is not clear. We have recently shown that the sensitivity of Kir2 channels to cholesterol depends on residues in the CD loop of the cytosolic domain of the channels with one of the mutations, L222I, abrogating cholesterol sensitivity of the channels completely. Here we show that in addition to Kir2 channels, members of other Kir subfamilies are also regulated by cholesterol. Interestingly, while similarly to Kir2 channels, several Kir channels, Kir1.1, Kir4.1 and Kir6.2Delta36 were suppressed by an increase in membrane cholesterol, the function of Kir3.4* and Kir7.1 was enhanced following cholesterol enrichment. Furthermore, we show that independent of the impact of cholesterol on channel function, mutating residues in the corresponding positions of the CD loop in Kir2.1 and Kir3.4*, inhibits cholesterol sensitivity of Kir channels, thus extending the critical role of the CD loop beyond Kir2 channels.     

Stochastic Ion Channel Gating in Dendritic Neurons: Morphology Dependence and Probabilistic Synaptic Activation of Dendritic Spikes

Neuronal activity is mediated through changes in the probability of stochastic transitions between open and closed states of ion channels. While differences in morphology define neuronal cell types and may underlie neurological disorders, very little is known about influences of stochastic ion channel gating in neurons with complex morphology. We introduce and validate new computational tools that enable efficient generation and simulation of models containing stochastic ion channels distributed across dendritic and axonal membranes. Comparison of five morphologically distinct neuronal cell types reveals that when all simulated neurons contain identical densities of stochastic ion channels, the amplitude of stochastic membrane potential fluctuations differs between cell types and depends on sub-cellular location. For typical neurons, the amplitude of membrane potential fluctuations depends on channel kinetics as well as open probability. Using a detailed model of a hippocampal CA1 pyramidal neuron, we show that when intrinsic ion channels gate stochastically, the probability of initiation of dendritic or somatic spikes by dendritic synaptic input varies continuously between zero and one, whereas when ion channels gate deterministically, the probability is either zero or one. At physiological firing rates, stochastic gating of dendritic ion channels almost completely accounts for probabilistic somatic and dendritic spikes generated by the fully stochastic model. These results suggest that the consequences of stochastic ion channel gating differ globally between neuronal cell-types and locally between neuronal compartments. Whereas dendritic neurons are often assumed to behave deterministically, our simulations suggest that a direct consequence of stochastic gating of intrinsic ion channels is that spike output may instead be a probabilistic function of patterns of synaptic input to dendrites.     

A Threshold Equation for Action Potential Initiation

In central neurons, the threshold for spike initiation can depend on the stimulus and varies between cells and between recording sites in a given cell, but it is unclear what mechanisms underlie this variability. Properties of ionic channels are likely to play a role in threshold modulation. We examined in models the influence of Na channel activation, inactivation, slow voltage-gated channels and synaptic conductances on spike threshold. We propose a threshold equation which quantifies the contribution of all these mechanisms. It provides an instantaneous time-varying value of the threshold, which applies to neurons with fluctuating inputs. We deduce a differential equation for the threshold, similar to the equations of gating variables in the Hodgkin-Huxley formalism, which describes how the spike threshold varies with the membrane potential, depending on channel properties. We find that spike threshold depends logarithmically on Na channel density, and that Na channel inactivation and K channels can dynamically modulate it in an adaptive way: the threshold increases with membrane potential and after every action potential. Our equation was validated with simulations of a previously published multicompartemental model of spike initiation. Finally, we observed that threshold variability in models depends crucially on the shape of the Na activation function near spike initiation (about −55 mV), while its parameters are adjusted near half-activation voltage (about −30 mV), which might explain why many models exhibit little threshold variability, contrary to experimental observations. We conclude that ionic channels can account for large variations in spike threshold.     

Charge selectivity of the designed uncharged peptide ion channel Ac-(LSSLLSL)3-CONH2.

Charge selectivity in ion channel proteins is not fully understood. We have studied charge selectivity in a simple model system without charged groups, in which an amphiphilic helical peptide, Ac-(Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2, forms ion channels across an uncharged phospholipid membrane. We find these channels to conduct both K+ and Cl-, with a permeability ratio (based on reversal potentials) that depends on the direction of the KCl concentration gradient across the membrane. The channel shows high selectivity for K+ when [KCl] is lowered on the side of the membrane that is held at a positive potential (the putative C-terminal side), but only modest K+ selectivity when [KCl] is lowered on the opposite side (the putative N-terminal side). Neither a simple Nernst-Planck electrodiffusion model including screening of the helix dipole potential, nor a multi-ion, state transition model allowing simultaneous cation and anion occupancy of the channel can satisfactorily fit the current-voltage curves over the full range of experimental conditions. However, the C-side/N-side dilution asymmetry in reversal potentials can be simulated with either type of model.     

Antibodies affect the ionic conductance of channels formed by amphotericin B in a lipid bilayer

For the first time poly- and monoclonal antibodies (class IgM) against the polyene antibiotic amphotericin B were obtained affecting the properties of a channel formed by the antibiotic and cholesterol in a lipid bilayer when amphotericin B was added to the solution at one (cis) side of the membrane. In the case of the symmetric distribution of cholesterol in the lipid bilayer, three molecules of monoclonal antibodies bind firmly to the channel at the trans-side of the membrane, thus strongly increasing the mean lifetime of the channel in the open state, and not changing practically the ion conductance of its open state. The antibodies did not alter the properties of these channels when added at the cis-side of the membrane as well as of the channels formed in the lipid bilayer when amphotericin B was added at both membrane sides. The antibodies obtained did not affect the conductance of channels in which amphotericin B and cholesterol were replaced with their analogs levorin and 5 alpha-androstan-3 beta-one, which points to a high specificity of the immunoglobulins isolated. When cholesterol was present only in the cis-monolayer of the lipid bilayer and was absent in the trans-monolayer, the same monoclonal antibodies when added at the trans-side of the membrane blocked the conductance of the channel formed by adding the antibiotic to the solution at the cis-side of the bilayer. The obtained evidence is of interest in elucidating the general features of interaction of antibodies with the ionic channels of cellular and model membranes.     

Cholesterol sensitivity of KIR2.1 is controlled by a belt of residues around the cytosolic pore

Kir channels play an important role in setting the resting membrane potential and modulating membrane excitability. A common feature of several Kir channels is that they are regulated by cholesterol. Yet, the mechanism by which cholesterol affects channel function is unclear. We recently showed that the cholesterol sensitivity of Kir2 channels depends on several CD-loop residues. Here we show that this cytosolic loop is part of a regulatory site that also includes residues in the G-loop, the N-terminus, and the connecting segment between the C-terminus and the inner transmembrane helix. Together, these residues form a cytosolic belt that surrounds the pore of the channel close to its interface with the transmembrane domain, and modulate the cholesterol sensitivity of the channel. Furthermore, we show that residues in this cluster are correlated with residues located in the most flexible region of the G-loop, the major cytosolic gate of Kir2.1, implying that the importance of these residues extends beyond their effect on the channel's cholesterol sensitivity. We suggest that the residues of the cholesterol sensitivity belt are critical for channel gating.     

Low molecular channel-former protein from the mitochondrial membrane

Individual low molecular weight protein component was isolated by gel-filtration method from the inner mitochondrial membrane. This membrane component increases the conductivity of bilayer lipid membranes (BLM) in the presence of K+ and Ca2+-ions. This phenomenon may be explained by the formation of single conductivity channels. The voltage - current characteristics of this channel is nonlinear, which may be the result of asymmetrical operation of the channel - former in the polarized membrane, in spite of equal concentration of protein on both sides of the membrane.     

Selectivity of latrotoxin channel in lipid bilayers]

It is established that high initial K-TEA and Ca2(+)-K+ selectivity of channel form by latrotoxin in lipid bilayers (LT-channel) may be reduced by lowering the pH value and by increasing electrolytes concentration of solution. It is suggested that LT-channel is water-filled pore cation selectivity which is defined by the electrostatic potential on the mouth of the channel, which is induced by the ionogenic group of toxin.     

A note on modeling mechano-chemical transduction with an application to a skin receptor

Strain-sensitive (also called stretch-sensitive) ionic channels are thought to be present in various mechanoreceptors. The gating of these channels is precipitated by mechanical strains, as opposed to the usual activation processes of changes in membrane potential or other ligands. Below we present a class of models for the strain-activated mechanism, compare our approach to one of Sachs and Lecar (1991), and apply the gating mechanism to a model of a specific mechanoreceptor, namely a Pacinian corpuscle neurite model. The simulation experiment suggests the activation energy of the channel depends linearly, rather than nonlinearly, on the (hoop) strain in the receptor membrane.     

An overview of trafficking and assembly of neurotransmitter receptors and ion channels (Review)

Ionotropic neurotransmitter receptors and voltage-gated ion channels assemble from several homologous and non-homologous subunits. Assembly of these multimeric membrane proteins is a tightly controlled process subject to primary and secondary quality control mechanisms. An assembly pathway involving a dimerization of dimers has been demonstrated for a voltage-gated potassium channel and for different types of glutamate receptors. While many novel C-terminal assembly domains have been identified in various members of the voltage-gated cation channel superfamily, the assembly pathways followed by these proteins remain largely elusive. Recent progress on the recognition of polar residues in the transmembrane segments of membrane proteins by the retrieval factor Rer1 is likely to be relevant for the further investigation of trafficking defects in channelopathies. This mechanism might also contribute to controlling the assembly of ion channels by retrieving unassembled subunits to the endoplasmic reticulum. The endoplasmic reticulum is a metabolic compartment studded with small molecule transporters. This environment provides ligands that have recently been shown to act as pharmacological chaperones in the biogenesis of ligand-gated ion channels. Future progress depends on the improvement of tools, in particular the antibodies used by the field, and the continued exploitation of genetically tractable model organisms in screens and physiological experiments.