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.

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.

Properties of the leakage current pathway: effects of the gadolinium ion on myelinated axons.

Voltage-clamp experiments on myelinated axons revealed that the trivalent gadoliniumion ion blocked the leakage current in a dose-dependent manner. The block showed 1:2 stoichiometry, and 50% reduction at 700 microM. Na+ and K+ currents were blocked at ten times lower concentration and showed 1:1 stoichiometry. Comparisons with biochemical studies suggest that the leakage current directly depends on a phospholipid pathway in contrast to the protein-channel dependent Na+ and K+ currents. The results may be explained by a leakage current pathway located at the interface between channel proteins and the lipid phase.

Single K(+)-channel currents under steady-state potential conditions in small hippocampal neurons.

Small cultured hippocampal neurons from rat embryos were studied with the patch-clamp technique. Single-channel currents from outside-out membrane patches were recorded under steady-state potential conditions. The most frequently found channel types were selective to K+ and showed conductances of about 30 and 80 pS in the range -20 to 0 mV. Two basic kinetic patterns were observed for the 80 pS channels. In one type, the fraction of time spent in open state increased with potential, and in the other type it decreased. For both types of 80 pS channel, the distribution of dwell times in the open state was well described by the sum of two exponentials while three exponentials sometimes were required for dwell times in the closed state. The time constants of the fitted exponentials could vary considerably during an experiment.

Impulses of variable amplitude recorded from cell-attached patches on small hippocampal neurons.

Small cultured neurons from embryonic rat hippocampus were investigated with the cell-attached configuration of the patch-clamp technique. The currents associated with spontaneous cellular impulse generation were studied. The positive peak current amplitudes varied by a factor larger than 2.5 for 11 of 12 cells quantitatively investigated, suggesting underlying action potentials of variable amplitude. A simple RC-network was found to describe the properties of the patch membrane, and was used to calculate the underlying potential time course. The action potentials computed showed a considerable variation in amplitude. The results suggest that the impulse variability previously analysed with the whole-cell technique was not caused by dialysis of the cell interior.

Ionic channels in the node of Ranvier are not modulated by cyclic adenosine monophosphate.

The effect of a cyclic adenosine monophosphate (cAMP) analogue on myelinated axons was studied. The lipophilic dibutyryl cAMP (dbcAMP) was applied to isolated frog axons under voltage clamp conditions. No effect on the ionic currents was observed for either external or internal application of dbcAMP. Nor was any effect of the phosphodiesterase inhibitor theophylline found. These results suggest that cAMP does not play an essential role for nerve impulse conduction in axons. This conclusion deviates from those of other studies.

Morphine activates calcium channels in cloned mouse neuroblastoma cell lines.

Membrane effects of morphine on cloned neuroblastoma cells (strains NG108-CC15 and N1E-115), were studied using intracellular recording and ion flux techniques. Morphine (10 microM) induced a depolarization that was affected by changes in the concentration of calcium, but not in sodium. Further, morphine induced an uptake of 45Ca2+ which was blocked by naloxone (1 microM). The simplest explanation for the findings is that morphine activates calcium channels via mu-receptors.

Ionic mechanisms of 3 types of functionally different neurons in the lamprey spinal cord.

Action potentials and afterpotentials were compared in giant interneurons, sensory dorsal cells and large intraspinal axons in the lamprey spinal cord. Afterpotentials of giant interneurons and dorsal cells consisted of two hyperpolarizing phases, an early and a late one, which were separated by a delayed depolarization. The afterpotentials of axons had a single hyperpolarizing phase also followed by a delayed depolarization. Tetraethyl ammonium chloride (TEA+) eliminated the early phase of the afterhyperpolarization in giant interneurons, only partially reduced the early phase in dorsal cells and did not affect the single phase of axons. The delayed depolarization of dorsal cells was attenuated by TEA+ but in axons it was unaltered. The heavy metal ions Mn2+ and Co2+ (2 mM) eliminated the late phase in giant interneurons but did not reduce the late phase in dorsal cells. The delayed depolarization remained in both types of cell in the presence of these ions. Action potentials of giant interneurons and dorsal cells, but not those of axons, were broadened by TEA+. The TEA-prolonged action potentials were narrowed by Mn2+ applied in combination with TEA+. The afterhyperpolarizations of all 3 cells were reduced by injection of negative current and enhanced by positive current. Repetitive stimulation resulted in summation of the afterhyperpolarization in giant interneurons and dorsal cells. The results suggest that different sets of potassium channels are responsible for the afterhyperpolarizations in each type of cell. In giant interneurons fast channels which are sensitive to TEA+ may underlie the early phase and slow channels activated by calcium entry may underlie the slow phase. The early phase of dorsal cells may be caused by two types of fast channel, one similar to that in giant interneurons and another less sensitive to external TEA+. This latter type may also cause the afterhyperpolarization in axons. Although calcium channels appear to contribute to the action potentials of giant interneurons and dorsal cells, the late phase of the latter neurons does not seem to be activated by calcium entry. The delayed depolarizations of the neurons appear to be due to an inward current which is not carried by calcium.

Single-channel and whole-cell currents in rat cerebellar granule cells.

The patch-clamp technique was used to study both whole-cell and single-channel currents in cultured rat cerebellar granule cells. In whole-cell recordings under voltage-clamp conditions 3 types of current were found; a transient inward sodium current and a transient and a sustained outward potassium current. Single-channel currents were recorded from both inside-out and outside-out membrane patches. Three types of potassium channel were identified; two non-inactivating channels with unit conductances of 160 and 60 pS and one inactivating channel of 20 pS. No calcium currents were detected.

Mechanisms of the tetrahydroaminoacridine effect on action potential and ion currents in myelinated axons.

9-Amino-1,2,3,4-tetrahydroacridine (THA) in the range of 10-300 microM was shown to prolong the action potential in myelinated nerve fibres of Xenopus laevis. Voltage-clamp experiments showed that THA, besides reducing the Na+ and the K+ current, modified the Na+ current inactivation and the K+ current activation. The effects were frequency dependent. Quantitative models were developed and used in computer simulations of the THA effect on the action potential. The computations showed that the observed effects on the ion currents were sufficient to explain the observed prolongation of the action potential. The models further suggest that THA binds to Na+ channels in an open state and from the axoplasmic side while it binds to K+ channels in a closed state. The findings suggest an explanation to some aspects of the clinical effects of THA on Alzheimer patients.

The mechanism of action of ketamine on the myelinated nerve membrane.

Ketamine is an intravenous anaesthetic that has been reported to react with a number of synaptic and non-synaptic receptors at both the spinal and supraspinal level. The present investigation was undertaken to analyse the effects of ketamine on the myelinated axon under voltage clamp conditions. Both sodium and potassium channels were affected. The effect may be described as mainly a reduction of the permeability constants. No effect on inactivation was observed. The effects were described by a first order binding to receptors within the ion channels that may be identical with the receptors for other anaesthetics. It was concluded from experiments with naloxone that no opiate receptors were involved in the axonal ketamine effects.

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.

Mechanisms of propofol action on ion currents in the myelinated axon of Xenopus laevis.

The effect of the intravenous anaesthetic, propofol (2,6-diisopropylphenol), was investigated on frog myelinated axons under voltage-clamp conditions. The effect, in the concentration range 60 microM to 10 mM, was a combination of (i) a negative shift of the steady state activation and inactivation curves for both Na+ and K+ currents (INa,IK), (ii) a voltage-independent block of INa, but not of IK, and (iii) a slowed time course of IK activation. The shift was dose-dependent and, at 1 mM, about -10 mV for the activation and -16 mV for the inactivation curves. The voltage-independent INa block showed 1:1 stoichiometry and 50% reduction at 2.7 mM. The slowed IK activation showed saturation at 1 mM with a doubled time to half steady state value. All the effects were only partially reversible and showed a complex time course at application and washing. The shift of potential dependence may be explained by a general effect on the membrane electric field. The findings suggest effects directly on channel proteins as well as on membrane lipids.

High concentrations of the neuroleptic remoxipride block voltage-activated Na+ channels in central and peripheral nerve membranes.

The actions of the neuroleptic compounds remoxipride, haloperidol and (-)-sulpiride on Na+ and K+ ion current flow were examined in rat CNS and frog PNS, using 86Rb+ ion flux and voltage-clamp techniques, respectively. By combining veratridine and high K(+)-evoked 86Rb+ efflux, it was determined that remoxipride blocked Na+ current flow in a concentration-dependent fashion in tissue pieces from either cerebral cortex or striatum (IC50 approximately 20 microM), leaving K+ current flow virtually unaffected. Similarly, haloperidol concentration dependently blocked Na+ current flow in both tissues (IC50 approximately 50 microM). (-)-Sulpiride did not have a significant effect. Direct actions of the compounds on voltage-gated Na+ and K+ channels were determined in voltage-clamp experiments. The findings confirmed the results of the ion flux measurements in that remoxipride (Kd approximately 300 microM) and haloperidol (Kd approximately 1.5 microM) reduced mainly the Na+ current, having little effect on the K+ current, whereas (-)-sulpiride did not have a measurable action. The relatively high concentrations of remoxipride or haloperidol needed to alter the Na+ current makes it unlikely that these actions are of importance at clinically relevant doses.

Effects of THA on ionic currents in myelinated axons of Xenopus laevis.

In voltage-clamp experiments with the myelinated nerve fibre of Xenopus laevis, 9-amino-1,2,3,4-tetrahydroacridine (THA) decreased both Na+ and K+ currents and shifted the steady state inactivation potential curve in a negative direction. The effects may be described as (a) a decrease of the permeability constant PNa, (b) a modified potential dependence of the inactivating system and (c) a decrease of PK. The Na+ system was affected more than the K+ system.

Graded action potentials in small cultured rat hippocampal neurons.

The 'all-or-nothing' principle has been central to neurobiology since the beginning of this century. We here demonstrate that action potentials in small cultured neurons from the hippocampus of rat embryos clearly depend on stimulus strength and thus deviate from this principle. We also demonstrate that similar stimulus-dependent action potentials can be predicted from computations based on voltage-clamp measurements. The findings suggest that amplitude modulation of the action potential may be a principle for information processing in the mammalian nervous system.

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.

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.

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.