Construction of a Ca(2+)-gated artificial channel by fusing alamethicin with a calmodulin-derived extramembrane segment.

Using native chemical ligation, we constructed a Ca(2+)-gated fusion channel protein consisting of alamethicin and the C-terminal domain of calmodulin. At pH 5.4 and in the absence of Ca(2+), this fusion protein yielded a burst-like channel current with no discrete channel conductance levels. However, Ca(2+) significantly lengthened the specific channel open state and increased the mean channel current, while Mg(2+) produced no significant changes in the channel current. On the basis of 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescent measurement, Ca(2+)-stimulated gating may be related to an increased surface hydrophobicity of the extramembrane segment of the fusion protein.

Benzimidazolium-based synthetic chloride and calcium transporters in bacterial membranes.

Herein, we present the first example of a benzimidazolium-based artificial transmembrane chloride transporter and a synthetic calcium ionophore that can regulate intracellular calcium concentrations in bacteria.

Designing calcium release channel inhibitors with enhanced electron donor properties: stabilizing the closed state of ryanodine receptor type 1.

New drugs with enhanced electron donor properties that target the ryanodine receptor from skeletal muscle sarcoplasmic reticulum (RyR1) are shown to be potent inhibitors of single-channel activity. In this article, we synthesize derivatives of the channel activator 4-chloro-3-methyl phenol (4-CmC) and the 1,4-benzothiazepine channel inhibitor 4-[-3{1-(4-benzyl) piperidinyl}propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (K201, JTV519) with enhanced electron donor properties. Instead of activating channel activity (~100 µM), the 4-methoxy analog of 4-CmC [4-methoxy-3-methyl phenol (4-MmC)] inhibits channel activity at submicromolar concentrations (IC(50) = 0.34 ± 0.08 µM). Increasing the electron donor characteristics of K201 by synthesizing its dioxole congener results in an approximately 16 times more potent RyR1 inhibitor (IC(50) = 0.24 ± 0.05 µM) compared with K201 (IC(50) = 3.98 ± 0.79 µM). Inhibition is not caused by an increased closed time of the channel but seems to be caused by an open state block of RyR1. These alterations to chemical structure do not influence the ability of these drugs to affect Ca(2+)-dependent ATPase activity of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase type 1. Moreover, the FKBP12 protein, which stabilizes RyR1 in a closed configuration, is shown to be a strong electron donor. It seems as if FKBP12, K201, its dioxole derivative, and 4-MmC inhibit RyR1 channel activity by virtue of their electron donor characteristics. These results embody strong evidence that designing new drugs to target RyR1 with enhanced electron donor characteristics results in more potent channel inhibitors. This is a novel approach to the design of new, more potent drugs with the aim of functionally modifying RyR1 single-channel activity.

Differential effects of temperature on E. coli and synthetic polyhydroxybutyrate/polyphosphate channels.

Complexes of poly-(R)-3-hydroxybutyrate and inorganic polyphosphate (PHB/polyP), isolated from the plasma membranes of Escherichia coli or prepared synthetically (HB(128)/polyP(65)), form Ca(2+)-selective ion channels in planar lipid bilayers that exhibit indistinguishable gating and conductance characteristics at 22 degrees C. Here we examine the gating and conductance of E. coli and synthetic PHB/polyP complexes in planar lipid bilayers as a function of temperature from 15 to 45 degrees C. E. coli PHB/polyP channels remained effectively open throughout this range, with brief closures that became more rare at higher temperatures. Conversely, as temperatures were gradually increased, the open probability of HB(128)/polyP(65) channels progressively decreased. The effect was fully reversible. Channel conductance exhibited three distinct phases. Below 25 degrees C, as PHB approached its glass temperature (ca. 10 degrees C), the conductance of both E. coli and synthetic channels remained at about the same level (95-105 pS). Between 25 degrees C and ca. 40 degrees C, the conductance of E. coli and synthetic channels increased gradually with temperature coefficients (Q(10)) of 1.45 and 1.42, respectively. Above 40 degrees C, E. coli channel conductance increased sharply, whereas the conductance of HB(128)/polyP(65) channels leveled off. The discontinuities in the temperature curves for E. coli channels coincide with discontinuities in thermotropic fluorescence spectra and specific growth rates of E. coli cells. It is postulated that E. coli PHB/polyP complexes are associated with membrane components that inhibit their closure at elevated temperatures.

Síntese de diidropirimidinonas e de uma biblioteca de análogos de vancomicina / Syntesis of dihidropyrimidonones and library of vancomycin analogues

A primeira parte desse trabalho refere-se à síntese de compostos do tipo diidropirimidinona (36), que säo de grande interesse farmacológico por serem moduladores de canal de cálcio. A síntese desses compostos foi feita através de uma reaçäo "one-pot" entre um aldeído (35), um "beta"-ceto-éster (34) e uréia (32), sob aquecimento por irradiaçäo de microondas. Os compostos foram obtidos com bons rendimentos, em apenas alguns minutos, sem a necessidade do uso de solventes ou catalisadores. De alguns dos compostos foram obtidos monocristais, analisados por cristalografia de raios-X, mostrando que os mesmos podem atuar como bloqueadores de canais de cálcio, pois apresentaram conformaçäo ideal para interaçäo...

Proof for a nonproteinaceous calcium-selective channel in Escherichia coli by total synthesis from (R)-3-hydroxybutanoic acid and inorganic polyphosphate.

Traditionally, the structure and properties of natural products have been determined by total synthesis and comparison with authentic samples. We have now applied this procedure to the first nonproteinaceous ion channel, isolated from bacterial plasma membranes, and consisting of a complex of poly(3-hydroxybutyrate) and calcium polyphosphate. To this end, we have now synthesized the 128-mer of hydroxybutanoic acid and prepared a complex with inorganic calcium polyphosphate (average 65-mer), which was incorporated into a planar lipid bilayer of synthetic phospholipids. We herewith present data that demonstrate unambiguously that the completely synthetic complex forms channels that are indistinguishable in their voltage-dependent conductance, in their selectivity for divalent cations, and in their blocking behavior (by La3+) from channels isolated from Escherichia coli. The implications of our finding for prebiotic chemistry, biochemistry, and biology are discussed.

Docking of verapamil in a synthetic Ca2+ channel: formation of a ternary complex involving Ca2+ ions.

The mechanism by which diverse drugs modulate voltage-dependent Ca2+ channels is ill-understood. We have approached this problem by examining the interaction of verapamil with a 97-residue synthetic channel peptide (SCP) that exhibits functional similarities to authentic L-type Ca2+ channels in terms of cation selectivity and permeation as well as interaction with channel-activating and blocking drugs (Grove et al. (1991) Proc. Natl. Acad. Sci. USA 88, 6418). Different possibilities of binding of verapamil inside the Ca(2+)-bound SCP were simulated using the Monte Carlo-with-energy-minimization method. In the optimal mode of the binding, verapamil adopted a folded conformation and fit snugly in the pore. The dimethoxyphenyl groups of the drug interacted with two Ca2+ ions coordinated to the acidic residues of SCP, thus forming a ternary complex of the drug, Ca2+, and channel. The isopropyl group of verapamil abetted a ring of four Ile residues constituting the putative SCP gate. The occlusion of this gate by verapamil in this manner was strikingly similar to that accomplished by the methyl group of dihydropyridine drugs. In conjunction with an earlier study on SCP bound to dihydropyridine drugs (Zhorov and Ananthanarayanan (1996) Biophys. J. 70, 22), our data suggest that, in general, drug modulation of SCP would involve the interaction of the ligands with the pore-bound Ca2+ and with the hydrophobic gate. In light of the functional similarity between SCP and L-type Ca2+ channel, it is likely that the latter would also interact with drugs in a similar fashion.