Proteasome system of protein degradation and processing

In eukaryotic cells, degradation of most intracellular proteins is realized by proteasomes. The substrates for proteolysis are selected by the fact that the gate to the proteolytic chamber of the proteasome is usually closed, and only proteins carrying a special “label” can get into it. A polyubiquitin chain plays the role of the “label”: degradation affects proteins conjugated with a ubiquitin (Ub) chain that consists at minimum of four molecules. Upon entering the proteasome channel, the polypeptide chain of the protein unfolds and stretches along it, being hydrolyzed to short peptides. Ubiquitin per se does not get into the proteasome, but, after destruction of the “labeled” molecule, it is released and labels another molecule. This process has been named “Ub-dependent protein degradation”. In this review we systematize current data on the Ub-proteasome system, describe in detail proteasome structure, the ubiquitination system, and the classical ATP/Ub-dependent mechanism of protein degradation, as well as try to focus readers’ attention on the existence of alternative mechanisms of proteasomal degradation and processing of proteins. Data on damages of the proteasome system that lead to the development of different diseases are given separately.     

Interactions Between Charged Residues in the Transmembrane Segments of the Voltage-sensing Domain in the hERG Channel

Studies on voltage-gated K channels such as Shaker have shown that positive charges in the voltage-sensor (S4) can form salt bridges with negative charges in the surrounding transmembrane segments in a state-dependent manner, and different charge pairings can stabilize the channels in closed or open states. The goal of this study is to identify such charge interactions in the hERG channel. This knowledge can provide constraints on the spatial relationship among transmembrane segments in the channel’s voltage-sensing domain, which are necessary for modeling its structure. We first study the effects of reversing S4’s positive charges on channel activation. Reversing positive charges at the outer (K525D) and inner (K538D) ends of S4 markedly accelerates hERG activation, whereas reversing the 4 positive charges in between either has no effect or slows activation. We then use the ‘mutant cycle analysis’ to test whether D456 (outer end of S2) and D411 (inner end of S1) can pair with K525 and K538, respectively. Other positive charges predicted to be able, or unable, to interact with D456 or D411 are also included in the analysis. The results are consistent with predictions based on the distribution of these charged residues, and confirm that there is functional coupling between D456 and K525 and between D411 and K538.     

Use of Nonelectrolytes Reveals the Channel Size and Oligomeric Constitution of the Borrelia burgdorferi P66 Porin

In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than ‘normal’ Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. The diameter of the entrance of the P66 channel was determined to be ≤1.9 nm and the channel has a central constriction of about 0.8 nm. The size of the channel appeared to be symmetrical as judged from one-sidedness of addition of NEs. Furthermore, the P66-induced membrane conductance could be blocked by 80–90% by the addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose to the aqueous phase in the low millimolar range. The analysis of the power density spectra of ion current through P66 after blockage with these NEs revealed no chemical reaction responsible for channel block. Interestingly, the blockage of the single-channel conductance of P66 by these NEs occurred in about eight subconductance states, indicating that the P66 channel could be an oligomer of about eight individual channels. The organization of P66 as a possible octamer was confirmed by Blue Native PAGE and immunoblot analysis, which both demonstrated that P66 forms a complex with a mass of approximately 460 kDa. Two dimension SDS PAGE revealed that P66 is the only polypeptide in the complex.     

Differences in abscisic acid concentration in roots and leaves of two young Olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) cultivars in response to water deficit

Differences in abscisic acid (ABA) accumulation between two olive cultivars were studied by enzyme-linked immunosorbent assay in roots and leaves, leaf water potential (Ψ_l), stomatal conductance (g _s) as well as photosynthetic rate (A) were also determined in well-watered (WW) and water-stressed (WS) plants of two olive cultivars ‘Chemlali’ and ‘Chetoui’. ‘Chemlali’ was able to maintain higher leaf CO₂ assimilation rate and leaf stomatal conductance throughout the drought cycle when compared with ‘Chetoui’. Furthermore, leaf water potential of ‘Chemlali’ decreased in lower extent than in Chetoui in response to water deficit. Interestingly, significant differences in water-stress-induced ABA accumulation were observed between the two olive cultivars and reflect the degree of stress experienced. Chemlali, a drought tolerant cultivar, accumulated lower levels of ABA in their leaves to regulate stomatal control in response to water stress compared to the drought sensitive olive cultivar ‘Chetoui’ which accumulated ABA in large amount.     

Lymphoma-selective antibody Lym-1 recognizes a discontinuous epitope on the light chain of HLA-DR10

 The selectivity of Lym-1 for malignant B lymphocytes makes this monoclonal antibody a promising candidate for the delivery of toxic agents to malignant B cells. The original immunogen used for the development of Lym-1 was Raji Burkitt’s lymphoma cell nuclei [Epstein A. L., Marder R. J., Winter J. N., Stathopoulos E., Chen F. M., Parker J. W., Taylor C. R. (1987) Cancer Res 47: 830]. The Lym-1 antigen was characterized at that time as a polymorphic HLA-DR variant. We prepared an affinity column using immobilized Lym-1 to isolate the Lym-1 antigen from Raji cell lysate. Immunological characterization of the immunoaffinity-purified Lym-1 antigen on Western blots led to the conclusion that the antigen is the β chain of HLA-DR10. This was confirmed by Edman sequencing of the isolated polypeptide chain. Western blots further show that the Lym-1 epitope is only recognized if the β chain disulfide bonds are intact. These results imply that Lym-1 binds a discontinuous epitope on the β chain of HLA-DR10. Received: 22 February 1996 / Accepted: 28 June 1996     

Statistical properties predicted by the ball and chain model of channel inactivation.

It has been proposed that part of a voltage gated channel is a tethered ball and that inactivation occurs when this wandering ball binds to a site in the channel. In order to be able to quantitatively test this model by comparison to experiments we developed analytical solutions and numerical simulations of the distribution of times it takes the ball to reach the binding site when the motion of the ball is random and when it is also influenced by a directed force. If the motion of the ball is one-dimensional, at long times this distribution is a single exponential with a rate constant that is inversely proportional to the square of the length of the chain and does not depend on the starting position of the ball. This dependence on the chain length is not significantly altered if there are short range electrical forces between the ball and its binding site. These predictions suggest that to confirm the validity of this model additional experiments should be done to more precisely determine the form of this distribution and its dependence on the length of the chain.     

Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels

The charge on the side chain of the internal pore residue lysine 519 (K519) of the Torpedo ClC-0 chloride (Cl-) channel affects channel conductance. Experiments that replace wild-type (WT) lysine with neutral or negatively charged residues or that modify the K519C mutant with various methane thiosulfonate (MTS) reagents show that the conductance of the channel decreases when the charge at position 519 is made more negative. This charge effect on the channel conductance diminishes in the presence of a high intracellular Cl- concentration ([Cl-]i). However, the application of high concentrations of nonpermeant ions, such as glutamate or sulfate (SO42-), does not change the conductance, suggesting that the electrostatic effects created by the charge at position 519 are unlikely due to a surface charge mechanism. Another pore residue, glutamate 127 (E127), plays an even more critical role in controlling channel conductance. This negatively charged residue, based on the structures of the homologous bacterial ClC channels, lies 4-5 A from K519. Altering the charge of this residue can influence the apparent Cl- affinity as well as the saturated pore conductance in the conductance-Cl- activity curve. Amino acid residues at the selectivity filter also control the pore conductance but mutating these residues mainly affects the maximal pore conductance. These results suggest at least two different conductance determinants in the pore of ClC-0, consistent with the most recent crystal structure of the bacterial ClC channel solved to 2.5 A, in which multiple Cl--binding sites were identified in the pore. Thus, we suggest that the occupancy of the internal Cl--binding site is directly controlled by the charged residues located at the inner pore mouth. On the other hand, the Cl--binding site at the selectivity filter controls the exit rate of Cl- and therefore determines the maximal channel conductance.     

pH-Modulation of Chloride Channels from the Sarcoplasmic Reticulum of Skeletal Muscle

The understanding of the role of cytoplasmic pH in modulating sarcoplasmic reticulum (SR) ion channels involved in Ca²⁺ regulation is important for the understanding of the function of normal and adversely affected muscles. The dependency of the SR small chloride (SCl) channel from rabbit skeletal muscle on cytoplasmic pH (pH _cis ) and luminal pH (pH _trans ) was investigated using the lipid bilayer-vesicle fusion technique. Low pH _cis 6.75–4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pH _cis 7.26–7.37 the channel mode is dominated by the conductance and kinetics of the main conductance state (65–75 pS) whereas at low pH _cis 6.75–4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5–40 pS). Similarly, low pH _trans 4.07, but not pH _trans 6.28, modified the activity of SCl channels. The effects of low pH _cis are pronounced at 10⁻³ and 10⁻⁴ m [Ca²⁺] _cis but are not apparent at 10⁻⁵ m [Ca²⁺] _cis , where the subconductances of the channel are already prominent. Low pH _cis -induced mode shift in the SCl channel activity is due to modification of the channel proteins that cause the uncoupling of the subconductance states. The results in this study suggest that low pH _cis can modify the functional properties of the skeletal SR ion channels and hence contribute, at least partly, to the malfunction in the contraction-relaxation mechanism in skeletal muscle under low cytoplasmic pH levels. Received: 20 May 1998/Revised: 24 September 1998     

Voltage-dependent channel formation by rods of helical polypeptides

Summary The voltage-dependence of channel formation by alamethicin and its natural analogues can be described by a dipole flip-flop gating model, based on electric field-induced transbilayer orientational movements of single molecules. These field-induced changes in orientation result from the large permanent dipole moment of alamethicin, which adopts -helical conformation in hydrophobic medium. It was, therefore, supposed that the only structural requirement for voltage-dependent formation of alamethicin-type channels might be a rigid lipophilic helical segment of minimum length.In order to test this hypothesis we synthesized a family of lipophilic polypeptides—Boc-(Ala-Aib-Ala-Aib-Ala) _n -OMe,n=1–4—which adopt -helical conformation forn=2–4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed qualitatively similar behavior. At low field strength of the membrane electric field these polypeptides induce a significant, almost voltage-independent increase of the bilayer conductivity. At high field strength, however, a strongly voltage-dependent conductance increase occurs similar to that observed with alamethicin. It results from the opening of a multitude of ion translocating channels within the membrane phase.The steady-state voltage-dependent conductance depends on the 8^th–9^th power of polypeptide concentration and involves the transfer of 4–5 formal elementary charges. From the power dependences on polypeptide concentration and applied voltage of the time constants in voltage-jump current-relaxation experiments, it is concluded that channels could be formed from preexisting dodecamer aggregates by the simultaneous reorientation of six formal elementary charges. Channels exhibit large conductance values of several nS, which become larger towards shorter polypeptide chain length. A mean channel diameter of 19 Å is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 -helical staves. Similar to experiments with the N-terminal Boc-derivative of alamethicin we did not observe the burst sequence of nonintegral conductance steps typical of natural (N-terminal Ac-Aib)-alamethicin. Saturation in current/voltage curves as well as current inactivation in voltage-jump current-relaxation experiments are found. This may be understood by assuming that channels are generated as dodecamers but, while reaching the steady state, reduce their size to that of an octamer or nonamer. We conclude that the overall behavior of these synthetic polypeptides is very similar to that of alamethicin. They exhibit the same concentration and voltage-dependences but lack the stabilizing principle of resolved channel states characteristic of alamethicin.     

Getting to Yes: Corporate Power and the Creation of a Psychopharmaceutical Blockbuster

In this paper, I analyze documentary evidence from a pharmaceutical company’s strategic marketing campaign to expand the sale of an antipsychotic medication beyond its conventional market. I focus on the role of the managerial function known as channel marketing, the task of which is to minimize friction, achieve coordination and add value in the distribution of the company’s products. However, the path to achieving these objectives is challenged because members of the marketing channel, or intermediaries, may not be contractual members of the channel; in fact they may have widely divergent goals or may even be hostile to the manufacturer’s efforts at control. This can be construed to be the case for physicians and others who are in the pharmaceutical manufacturer’s distribution channel but not of it. Their views and actions must somehow be brought into alignment with the manufacturer’s goals. This paper seeks to show part of the process from the manufacturer’s strategic standpoint, in which potential dissenters are incorporated into the pharmaceutical company distribution channel. The routinization of this incorporation results in the diminishment of psychiatry’s professional autonomy by means of what is—paradoxically to them, but not to a student of marketing—a competitive threat. The paper concludes with a discussion of corporate power.

The Hormonal Control of Uterine Luminal Fluid Secretion and Absorption

GABA_A receptors composed of α, β and γ subunits display a significantly higher single-channel conductance than receptors comprised of only α and β subunits. The pore of GABA_A receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at the 6′, 10′ and 13′ positions. At the 2′ position, however, a polar residue is present in the γ subunit but not the α or β subunits. As residues at the 2′, 6′ and 10′ positions are exposed in the open channel and as such polar channel-lining residues may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and the kinetic properties of wild-type α1β1 and α1β1γ2S receptors with two mutant receptors, αβγ(S2′A) and αβγ(S2′V). We found that the single-channel conductance of both mutant αβγ receptors was significantly decreased with respect to wild-type αβγ, with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant αβγ receptors remained larger than wild-type αβ channels. This reduction in the conductance of mutant αβγ receptors was observed at depolarized potentials only (E_Cl = −1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the γ2′ residue. The substitutions at the γ2′ serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study show that residues at the 2′ position in M2 of the γ subunit affects both single-channel conductance and receptor kinetics.     

Modulation of proton-induced current fluctuations in the human nicotinic acetylcholine receptor channel

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that switches upon activation from a closed state to a full conducting state. We found that the mutation δ S268K, located at 12′ position of the second transmembrane domain of the δ subunit of the human nAChR generates a long-lived intermediate conducting state, from which openings to a wild-type like conductance level occur on a submillisecond time scale. Aiming to understand the interplay between structural changes near the 12′ position and channel gating, we investigated the influence of various parameters: different ligands (acetylcholine, choline and epibatidine), ligand concentrations, transmembrane voltages and both fetal and adult nAChRs. Since sojourns in the high conductance state are not fully resolved in time, spectral noise analysis was used as a complement to dwell time analysis to determine the gating rate constants. Open channel current fluctuations are described by a two-state Markov model. The characteristic time of the process is markedly influenced by the ligand and the receptor type, whereas the frequency of openings to the high conductance state increases with membrane hyperpolarization. Conductance changes are discussed with regard to reversible transfer reaction of single protons at the lysine 12′ side chain.     

Transport-active organs within the ’ano-genital’ capsule of Craterostigmus tasmanianus (Chilopoda, Craterostigmomorpha)

 In Craterostigmus tasmanianus, first results of the cellular organization of anal organs within the ’ano-genital’ capsule are presented. Each valve of the ’ano-genital’ capsule bears four pore fields ventrally, each of them consisting of several pore openings of the anal organs. The pores lead into a cuticle-lined pore channel, the base of which is surrounded by a single-layered epithelium that is composed of three different cell types. The main epithelium consists of radially arranged transport-active cells surrounded by exocrine cells, and the cells of the pore channel. The cells of the transporting epithelium show deep invaginations of the apical and basal cell surfaces and plasmalemma-mitochondrial complexes. These cells are covered by a specialized cuticle with a prominent subcuticle. Exocrine glands secrete a mucous layer on the cuticle of the main epithelium. The type of anal organ present in Craterostigmus tasmanianus shows similarities to coxal and anal organs found in other Pleurostigmophora in the chilopods. The possible function of the anal organs in uptaking water vapour is discussed. It is appropriate to call the organs within the ’ano-genital’ capsule of Craterostigmus tasmanianus ”anal organs”, as components of the genital segments are not involved. Accepted: 17 November 1996     

Isoenzyme markers in varietal identification of banana

Summary Salt-soluble polypeptide and a few isozymes were profiled to identify banana cultivars available in Andamans, India. Salt-soluble polypeptide profile was found to be inappropriate in cultivar identification However, isozymes such as peroxidase could differentiate ‘Jungli kela’, ‘Tissue Cultured Dwarf Cavendish’ (TCDC), ‘Lal kela’, ‘Rajbel’, and ‘Baratang wild’, while esterase identified all the cultivars except ‘Rajbel’ and ‘Tarkari kela’. The latter two cultivars could be identified with the use of malate dehydrogenase (MDH) and peroxidase profiles, MDH portrayed cultivar-specific distinct banding pattern in ‘Khatta Champa’, ‘Tarkari kela’, and ‘Baratang wild’, ‘China kela’ could be identified easily by superoxide dismutase (SOD). Amongst four isozymes, esterase was found to be most efficient in identifying eight cultivars amongst 10; bence this isozyme may be used often as a marker for cultivar identification of banana.     

Mutations in the M4 Domain of the Torpedo californica Nicotinic Acetylcholine Receptor Alter Channel Opening and Closing

We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type α418Cys residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold, respectively. The mutation of a homologous cysteine in the β subunit (β447Trp) had similar but smaller effects on mean open time. Coexpression of α418Trp and β447Trp had the largest effect on channel open time, increasing mean open time 58-fold. No changes in conductance or ion selectivity were detected for any of the single subunit amino acid substitutions tested. However, the coexpression of the α418Trp and β447Trp mutated subunits also produced channels with at least two additional conductance levels. Block by acetylcholine was apparent in the current records from α418Trp mutants. Burst analysis of the α418Trp mutations showed an increase in the channel open probability, due to a decrease in the apparent channel closing rate and a probable increase in the effective opening rate. Our results show that modifications in the primary structure of the α- and β subunit M4 domain, which are postulated to be at the lipid-protein interface, can significantly alter channel gating, and that mutations in multiple subunits act additively to increase channel open time. Received: 27 September 1996/Revised: 28 January 1997     

The role of protein kinase C in the establishment of the mechanism of vasopressin antidiuretic action in the rat kidney during mammalian postnatal development

The kidney of immaturely born mammals in early postnatal development is insensitive to the effect of the antidiuretic hormone, vasopressin. It has been demonstrated that water permeability of the epithelial cells in the collecting ducts of a rat kidney increases during development; in this process, the response to desmopressin, an agonist of vasopressin V2 receptors, appears at the age of 20 days. The observed increase in water permeability is connected with an increased content of the water channel proteins aquaporins AQP2 and AQP3 in the plasma membrane. The calcium-dependent protein kinase C isoforms are the likely components of the vasopressin signal transduction and are possibly involved in the mechanisms underlying the maturation of sensitivity to this hormone. The contents of three protein kinase C isoforms (α,δ, and ζ) in rats at different periods of their postnatal development were estimated using Western blot hybridization. It has been shown that the contents of protein kinase C isoforms α and δ increase with development, whereas the content of isoform ζ remains constant. The most likely participant of the mechanism providing for maturation of the cell’s hormonal competence for vasopressin is the calcium-dependent protein kinase Cα, because it’s content in the plasma membrane is maximal on days 20–24, which coincides with the time when the vasopressin action appears.     

Differentiation-dependent changes in the membrane properties of fiber cells isolated from the rat lens

Impedance measurements in whole lenses showed that lens fiber cells possess different permeability properties to the epithelial cells from which they differentiate. To confirm these observations at the cellular level, we analyzed the membrane properties of fiber cells isolated in the presence of the nonselective cation channel inhibitor Gd³⁺. Isolated fiber cells were viable in physiological [Ca²⁺] and exhibited a range of lengths that reflected their stage of differentiation. Analysis of a large population of fiber cells revealed a subgroup of cells whose conductivity matched values measured in the whole lens (1). In this group of cells, membrane resistance, conductivity, and reversal potential all varied with cell length, suggesting that the process of differentiation is associated with a change in the membrane properties of fiber cells. Using pharmacology and ion substitution experiments, we showed that newly differentiated fiber cells (<150 µm) contained variable combinations of Ba²⁺-and tetraethylammonium-sensitive K⁺ currents. Longer fiber cells (150–650 µm) were dominated by a lyotropic anion conductance, which also appears to plays a role in the intact lens. Longer cells also exhibited a low-level, nonselective conductance that was eliminated by the replacement of extracellular Na⁺ with N-methyl-D-glucamine, indicating that the lens contains both Gd³⁺-sensitive and -insensitive nonselective cation conductances. Fiber cell differentiation is therefore associated with a shift in membrane permeability from a dominant K⁺ conductance(s) toward larger contributions from anion and nonselective cation conductances as fiber cells elongate. electrophysiology; potassium channel; anion channel; nonselective cation channel     

Two inward K+ channels in the xylem parenchyma cells of barley roots are regulated by G-protein modulators through a membrane-delimited pathway

In order to study the mechanism and regulation of K⁺ resorption from the xylem by the cells that border the xylem vessels (the xylem parenchyma cells), K⁺ inward-rectifying channels (KIRCs) in the plasma membrane of xylem parenchyma cells from Hordeum vulgare L. cv. Apex were studied using the patch-clamp technique. In the inside-out configuration, three different types of K⁺ channel and a further K⁺ conductance could be identified. Two of these channels, named KIRC1 and KIRC2, were activated by guanosine 5′-[β,γ-imido]triphosphate (Gpp(NH)p; 150 μM), a non-hydrolyzable derivative of GTP, indicating that channel activity was up-regulated by G-proteins; modulation of channel activity occurred via a membrane-delimited pathway, since the effect could be demonstrated in cell-free patches. At 100 mM external K⁺, KIRC1 had a conductance of 8 pS. There was no effect of ATP on channel activity. Likewise, addition of 150 μM guanosine 5′-[β-thio]diphosphate (GDPβS) or adenosine 5′-[γ-thio]triphosphate (ATPγS) failed to activate KIRC1, indicating nucleotide specificity of the effect. A second K⁺ channel, activated by Gpp(NH)p (KIRC2) with gating properties clearly different from the first one was less frequently observed. Four different substates could be identified; the main level had a conductance of about 2 pS. Gating below the Nernst potential of K⁺ (E_K) was voltage-independent. The channel closed at potentials more positive than E_K. A third, hyperpolarization-activated K⁺ channel, KIRC3, with a low open probability was encountered in inside-out patches. It had a conductance of 45 pS in 100 mM K⁺. Channel activity was not affected by the addition of G-protein modulators. Moreover, slowly activating inward currents carried by K⁺ were recorded in several patches that are ascribed to a `subpicosiemens conductance'. Neither GDPβS nor Gpp(NH)p appeared to have an effect on the currents. Whole-cell measurements with these G-protein modulators included in the pipette solution were in general agreement with the results obtained on cell-free patches. A statistical evaluation revealed that time-dependent inward currents were larger when the G-protein activator Gpp(NH)p was included in the pipette medium compared to measurements with the inhibitor GDPβS. With the GTP analogue, an additional instantaneous component was elicited that was ascribed to KIRC2 activity. Data are discussed with respect to the putative role of G-proteins in conveying hormonal signals. Regulation by G-protein may either serve to fine-tune K⁺ uptake by xylem parenchyma cells or to initiate depolarization, followed by salt-efflux through depolarization-activated cation and anion channels. Received 11 October 1996 / Accepted: 21 April 1997     

Computer simulation of polypeptides in a confinement

A coarse-grained model of polypeptide chains confined in a slit formed by two parallel impenetrable surfaces was studied. The chains were flexible heteropolymers (polypeptides) built of two kinds of united atoms—hydrophobic and hydrophilic. The positions of the united atoms were restricted to the vertices of a [310] lattice. The force field consisted of a rigorous excluded volume, a long-distance potential between a pair of amino-acid residues and a local preference for forming secondary structure (helices). The properties of the chains were studied at a wide range of temperatures from good to bad solvent conditions. Monte-Carlo simulations were carried out using the algorithm based on the chain’s local changes of conformation and employing the Replica Exchange technique. The influence of the chain length, the distances between the confining surfaces, the temperature and the force field on the dimension and the structure of chains were studied. It was shown that the presence of the confinement chain complicates the process of the chain collapse to low-temperature structures. For some conditions, one can find a rapid decrease of chain size and a second transition indicated by the rapid decrease of the total energy of the system. Figure A scheme of a polypeptide chain built on a [310] lattice and confined to a slit formed by a pair of parallel impenetrable surfaces Proceedings of “Modeling Interactions in Biomolecules II,” Prague, September 5th–9th, 2005.     

Temperature Sensitivity of the K+ Channel of Chara. A Thermodynamic Analysis

Temperature sensitivity of the K⁺ channel of Chara cytoplasmicdroplets has been characterized by means of the patch-clamptechnique. The activity of the channel was recorded in inside-outpatches over a range of temperatures (3°C to 25°C).An increment in the unitary channel conductance and a decreasein the probability of channel opening was found as temperatureaugmented. This could be explained by the combined effect ofa reduction in the mean open duration and an increase in theclosed times (Q₁₀ values of 0.7 and 1.1–1.6 respectively).Eyring's transition state theory was applied to the thermodynamicanalysis of conductance and kinetics of the K⁺ channel. Thevalues obtained for the activation enthalpy and entropy werecompared with, and found to be similar to, those reported forvoltage-dependent K⁺ channels in animal cells. The relativeinsensitivity of channel conductance to temperature (activationenthalpy of 2.4 kcal mol^–1) suggests that ions traversethe pore by diffusion. Channel closure appears to have the highestenergetic requirements (activation enthalpy of 6.4 kcal mol^–1).The channel closing rate, , exhibits a less negative entropicchange (–22.54 cal K^–1 mol^–1), which wouldprovide the driving force for stabilizing the closed configurationof the channel as the temperature increases. (Received September 16, 1993; Accepted December 3, 1993)