Role of cooperativity in protein folding and protein mosaic assemblage relevance for protein conformational diseases.

Biological systems are organized in intricate and highly structured networks with hierarchies and multiple scales. Cells can be considered as "meso-scale level" systems placed between the "macro-scale level" (systems of cellular networks) and the "micro-scale level" (systems of molecular networks). In fact, cells represent complex biochemical machineries made by networks of molecules connected by biochemical reactions. Thus, the brain should be studied as a system of "networks of networks". Recently, the existence of a Global Molecular Network (GMN) enmeshing the entire CNS was proposed. This proposal is based on the evidence that the extra-cellular matrix is a dynamic molecular structure capable of storing and releasing signals and of interacting with receptors and proteins on the cell membranes. Proteins have a special role in molecular networks since they can be assembled into high-order molecular complexes, which have been defined as Protein Mosaics (PM). Protein monomers in a PM (the "tesserae" of the mosaic) can interact via classical and non-classical cooperativity behaviour involving allosteric interactions. In the present paper, new features of allostery and cooperativity for protein folding, assemblage and topological features of PM will be discussed. Against this background, alterations in PM via allosteric modulations and non-classical cooperativity mechanisms may lead to protein aggregates like beta amyloid fibrils. Such aggregates cause pathological changes in the GMN structure and function leading to neurodegenerative diseases such as Alzheimer's disease. Thus, a novel view of the so called Protein Conformational Diseases (PCD) is proposed.

Single residue (K332A) substitution in Kir6.2 abolishes the stimulatory effect of long-chain acyl-CoA esters: indications for a long-chain acyl-CoA ester binding motif.

AIMS/HYPOTHESIS: The pancreatic beta cell ATP-sensitive potassium (K(ATP)) channel, composed of the pore-forming alpha subunit Kir6.2, a member of the inward rectifier K+channel family, and the regulatory beta subunit sulfonylurea receptor 1 (SUR1), a member of the ATP-binding cassette superfamily, couples the metabolic state of the cell to electrical activity. Several endogenous compounds are known to modulate K(ATP) channel activity, including ATP, ADP, phosphatidylinositol diphosphates and long-chain acyl coenzyme A (LC-CoA) esters. LC-CoA esters have been shown to interact with Kir6.2, but the mechanism and binding site(s) have yet to be identified. MATERIALS AND METHODS: Using multiple sequence alignment of known acyl-CoA ester interacting proteins, we were able to identify four conserved amino acid residues that could potentially serve as an acyl-CoA ester-binding motif. The motif was also recognised in the C-terminal region of Kir6.2 (R311-332) but not in SUR1. RESULTS: Oocytes expressing Kir6.2DeltaC26 K332A repeatedly generated K(+)currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 mumol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2DeltaC26), Kir6.2DeltaC26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2DeltaC26 K332A and SUR1 revealed slightly increased activation by palmitoyl-CoA ester but significantly decreased activation by the acyl-CoA esters compared with the wild-type K(ATP) channel and Kir6.2DeltaC26+SUR1. Computational modelling, using the crystal structure of KirBac1.1, suggested that K332 is located on the intracellular domain of Kir6.2 and is accessible to intracellular modulators such as LC-CoA esters. CONCLUSIONS/INTERPRETATION: These results verify that LC-CoA esters interact at the pore-forming subunit Kir6.2, and on the basis of these data we propose an acyl-CoA ester binding motif located in the C-terminal region.

Channel density regulation of firing patterns in a cortical neuron model.

Modifying the density and distribution of ion channels in a neuron (by natural up- and downregulation or by pharmacological intervention or by spontaneous mutations) changes its activity pattern. In this investigation we analyzed how the impulse patterns are regulated by the density of voltage-gated channels in a neuron model based on voltage-clamp measurements of hippocampal interneurons. At least three distinct oscillatory patterns, associated with three distinct regions in the Na-K channel density plane, were found. A stability analysis showed that the different regions are characterized by saddle-node, double-orbit, and Hopf-bifurcation threshold dynamics, respectively. Single, strongly graded action potentials occur in an area outside the oscillatory regions, but less graded action potentials occur together with repetitive firing over a considerable range of channel densities. The relationship found here between channel densities and oscillatory behavior may partly explain the difference between the principal spiking patterns previously described for crab axons (class 1 and 2) and cortical neurons (regular firing and fast spiking).

Na channel kinetics: developing models from non-stationary current fluctuations by analytic methods.

In a previous study, we analyzed Na current fluctuations in myelinated axons from Xenopus laevis under voltage clamp conditions. The statistical properties were analyzed in terms of covariance functions for consecutive time intervals of varying duration during the pulse step. The underlying channel kinetics was analyzed by performing stochastic simulations of published Na channel models and calculating corresponding covariance functions. None of the models explained the fluctuation results. We therefore developed a novel minimal Na channel model that satisfactorily described the results. In the present paper, we extend the analysis and specify the possible models explaining the experimental data by using analytical methods. We derive general relations between the experimental data, including the covariance functions, and the rate constants of specific one-open-state models. A general feature of these models is that they comprise an inactivation step from the first closed state and a relatively low backward rate from the open state. This is in accordance the minimal model inferred from numerical stochastic calculations in the previous study.

Non-stationary fluctuation analysis of the Na current in myelinated nerve fibers of Xenopus laevis: experiments and stochastic simulations.

Na current fluctuations under voltage-clamp conditions during pulse steps in the potential range from -65 to -30 mV were measured in myelinated nerve fibers of Xenopus laevis. The covariance functions for four consecutive 1 ms intervals were calculated. The time courses of the covariance functions were well fitted with monoexponential functions with time constants between 0.5 and 3 ms, larger at the end of the pulse and larger at more positive potentials. To analyze the underlying channel kinetics we simulated current fluctuations at a step to -35 mV of eight published Na channel models and calculated corresponding covariance functions. None of the models did explain the experimental fluctuation results. We therefore developed a new Na channel model that satisfactorily described the results. Features that distinguished this model from the other tested ones were a slower deactivation rate, and an inactivation transition directly from a closed state.

NMDA and glycine regulate the affinity of the Mg2+-block site in NR1-1a/NR2A NMDA receptor channels expressed in Xenopus oocytes.

NMDA receptors are glutamate-regulated ion channels of critical importance for many neurophysiological and neuropathological processes. Mg2+ blocks the NMDA receptor by binding to the channel pore with an apparent affinity that depends on the membrane potential. We have investigated the effect of NMDA and the required co-agonist glycine on the affinity of the Mg2+ block site in NR1-1a/NR2A NMDA receptors expressed in Xenopus oocytes. We found that NMDA and glycine increase the IC50 value of the Mg2+-block site at pH 7.4 and in the presence of physiological concentration of Ca2+. The increase the IC50 value may correspond to a decrease in Mg2+-block affinity. This effect may result in an increased influx of Ca2+, and this influx may constitute up to a third of the total Ca2+ influx induced by NMDA. At high pH, or at low concentrations of Ca2+, NMDA and glycine have an opposite effect and instead decreased the IC50 value of the Mg2+-block. These results indicate that glutamate and glycine can regulate the affinity of the Mg2+-block site. This effect may have implications for the understanding the role of NMDA receptors both under physiological and pathophysiological conditions.

Localization of the extracellular end of the voltage sensor S4 in a potassium channel.

The opening and closing of the pore of voltage-gated ion channels is the basis for the nervous impulse. These conformational changes are triggered by the movement of an intrinsic voltage sensor, the fourth transmembrane segment, S4. The central problem of how the movement of S4 is coupled to channel opening and where S4 is located in relation to the pore is still unsolved. Here, we estimate the position of the extracellular end of S4 in the Shaker potassium channel by analyzing the electrostatic effect of introduced charges in the pore-forming motif (S5-S6). We also present a three-dimensional model for all transmembrane segments. Knowledge of this structure is essential for the attempts to understand how voltage opens these channels.

Role of individual surface charges of voltage-gated K channels.

Fixed charges on the extracellular surface of voltage-gated ion channels influence the gating. In previous studies of cloned voltage-gated K channels, we found evidence that the functional surface charges are located on the peptide loop between the fifth transmembrane segment and the pore region (the S5-P loop). In the present study, we determine the role of individual charges of the S5-P loop by correlating primary structure with experimentally calculated surface potentials of the previously investigated channels. The results suggest that contributions to the surface potential at the voltage sensor of the different residues varies in an oscillating pattern, with the first residue of the N-terminal end of the S5-P loop, an absolutely conserved glutamate, contributing most. An analysis yields estimates of the distance between the residues and the voltage sensor, the first N-terminal residue being located at a distance of 5-6 A. To explain the results, a structural hypothesis, comprising an alpha-helical N-terminal end of the S5-P loop, is presented.

Mechanisms of bupivacaine action on Na+ and K+ channels in myelinated axons of Xenopus laevis.

The local anaesthetic bupivacaine has recently been proposed to inhibit Na+ channels indirectly by making the resting potential less negative. To test this hypothesis we analysed the effects of bupivacaine on voltage and current clamped nodes of Ranvier. Contrary to the hypothesis, the leak current and the resting potential were unaffected. The Na+ and K+ channels were, however, affected at relatively low concentrations (33 microM). Steady-state activation curves were decreased without notable shift effects, whereas the Na+ inactivation curve was decreased and shifted in negative direction. The effect on the Na+ current was tentatively explained by a single-site, state-dependent binding model (Kd = 44 microM), while that on the K+ current was explained by two population-specific mechanisms, one open-state dependent (Kd = 550 microM) and one state independent (Kd = 59 microM). The binding stoichiometry was higher than 1:1 for the main sites of action. In conclusion, bupivacaine exerts its main anaesthetic action on myelinated nerve axons by a direct modification of Na+ channels.

The functional surface charge density of a fast K channel in the myelinated axon of Xenopus laevis.

The action of Mg2+ on the putative xKv1.1 channel in the myelinated axon of Xenopus laevis was analyzed in voltage clamp experiments. The main effect was a shift in positive direction of the open probability curve (16 mV at 20 mM Mg2+), calculated from measurements of the instantaneous current at Na reversal potential after 50-100 msec steps to different potentials. The shift was measured at an open probability level of 25% to separate it from shifts of other K channel populations in the nodal region. The results could be explained in terms of screening effects on fixed charges located on the surface of the channel protein. Using the Grahame equation the functional charge density was estimated to -0.45 e nm-2. Analyzing this value, together with previously estimated values from other K channels, with reference to the charge of different extracellular loops of the channel protein, we conclude that the loop between the transmembrane S5 segment and the pore forming P segment determines the functional charge density of voltage-gated K channels.

Divalent cation effects on the Shaker K channel suggest a pentapeptide sequence as determinant of functional surface charge density.

The effects of the divalent cations strontium and magnesium on Shaker K channels expressed in Xenopus oocytes were investigated with a two-electrode voltage-clamp technique. 20 mM of the divalent cation shifted activation (conductance vs. potential), steady-state inactivation and inactivation time constant vs. potential curves 10-11 mV along the potential axis. The results were interpreted in terms of the surface charge theory, and the surface charge density was estimated to be -0.27 e nm-2. A comparison of primary structure data and experimental data from the present and previous studies suggests that the first five residues on the extracellular loop between transmembrane segment 5 and the pore region constitutes the functional surface charges. The results further suggest that the surface charge density plays an important role in controlling the activation voltage range.

On the coevolution of cognition and consciousness.

In this article it is argued that an evolutionary perspective leads to the view that adaptation and learning is a widespread and old property of living organisms, even as old as life itself. Cognition, defined as knowledge processing mediated by a centralised nervous system, is suggested mainly to be based on the same principles as non-neural adaptive processes. The emergence of conscious cognition, however, is seen as a major transition in the evolution of life, although it appears in different degrees and at various stages in evolution. Both cognition and consciousness depend on the organisation and complexity of the organism, primarily with regard to the nervous system. Computational and neurophysiological approaches are discussed, in particular some experimental attempts to determine anatomical, physiological and physical correlates to consciousness. It is argued that an evolutionary perspective suggests an interactionistic solution to the mind-brain problem, i.e. the question of subjective experience. In an interactionistic perspective consciousness can be understandable as a biological phenomenon. It can be regarded as a driving force in evolution, amplifying and improving the adaptive and cognitive processes of an organism.

Tail currents in the myelinated axon of Xenopus laevis suggest a two-open-state Na channel.

Na tail currents in the myelinated axon of Xenopus laevis were measured at -70 mV after steps to -10 mV. The tail currents were biexponential, comprising a fast and a slow component. The time constant of the slow tail component, analyzed in the time window 0.35-0.50 ms, was independent of step duration, and had a value of 0.23 ms. The amplitude, extrapolated back to time 0, varied, however, with step duration. It reached a peak after 0.7 ms and inactivated relatively slowly (at 2.1 ms the absolute value was reduced by approximately 30%). The amplitude of the fast component, estimated by subtracting the amplitude of the slow component from the calculated total tail current amplitude, reached a peak (three times larger than that of the slow component) after 0.5 ms and inactivated relatively fast (at 2.1 ms it was reduced by approximately 65%). The results were explained by a novel Na channel model, comprising two open states bifurcating from a common closed state and with separate inactivating pathways. A voltage-regulated use of the two pathways explains a number of findings reported in the literature.

Surface Charges of K channels. Effects of strontium on five cloned channels expressed in Xenopus oocytes.

The effects of strontium (Sr2+; 7-50 mM) on five different cloned rat K channels (Kv1.1, Kv1.5, Kv1.6, Kv2.1, and Kv3.4), expressed in oocytes of Xenopus laevis, were investigated with a two-electrode voltage clamp technique. The main effect was a shift of the Gk(V) curve along the potential axis, different in size for the different channels. Kv1.1 was shifted most and Kv3.4 least, 21 and 8 mV, respectively, at 50 mM. The effect was interpreted in terms of screening of fixed surface charges. The estimated charge densities ranged from -0.37 (Kv1.1) to -0.11 (Kv3.4) e nm-2 and showed good correlation with the total net charge of the extracellularly located amino acid residues of the channel as well as with the charge of a specific region (the loop between the S5 segment and the pore forming segment). The estimated surface potentials were found to be linearly related to the activation midpoint potential, suggesting a functional role for the surface charges.

Spontaneous signalling in small central neurons: mechanisms and roles of spike-amplitude and spike-interval fluctuations.

Spontaneous brain activity is essential for normal brain function. We are studying spontaneous activity in hippocampus at several complexity levels: at the microscopic level by analyzing the role of ion channels, at the mesoscopic level by analyzing the neuronal impulse activity, and at the macroscopic level by computational studies of mean electric fields of cortical network models. We have focused on the role of a subset of hippocampal neurons in the rat--neurons of small size (diameter < 10 microns). The analysis of spontaneous impulse trains in these neurons, both isolated and in slices, show (i) that impulses vary in amplitude, the magnitude depending on the input signal, suggesting that the amplitude variability may play a role in the information processing of the brain, and (ii) that single ion channel events can trigger neuronal impulses, suggesting that these neurons can function as cellular random generators. The possible role of random generators are investigated by simulating spontaneous activity in a cortical network model, based on a simplified description of the architecture of the CA1 area of hippocampus. The simulations show that such random generators can induce synchronous oscillations in cortical networks. These findings highlight the role of microfluctuations for the global macroactivity of the brain, and stress the importance of the study of channel kinetics for brain physiology.

Vertical information flow in the brain: on neuronal micro events and consciousness.

The consciousness problem and its relation to theories of spontaneous brain activity are discussed. It is argued that an evolutionary perspective suggests an interactionist solution of the consciousness problem, i.e., mental events interact with physical brain events. A specification of the physical events assumed to be associated with mental events is proposed to make the discussion of mind-brain theories more fruitful. An interactionist solution of the consciousness problem requires random spontaneous brain activity. A mechanism for such activity is proposed on the basis of experimental findings. Some new results from patch-clamp experiments on intact tissue (the hippocampal slice preparation) are presented. The results indicate that single channel events may cause random spontaneous neuronal activity, illustrating the phenomenon of micro events inducing macro or global brain events, and also the central theme of vertical information flow. The findings highlight the importance of understanding the stochastic nature of channel gating.