Tajib A. Mirzabekov

Membrane channel formation by antimicrobial protegrins.

Protegrins are small, arginine- and cysteine-rich, beta-sheet peptides with potent activity against bacteria, fungi, and certain enveloped viruses. We report that protegrins form weakly anion-selective channels in planar phospholipid bilayers, induce potassium leakage from liposomes and form moderately cation-selective channels in planar lipid membranes that contain bacterial lipopolysaccharide. The disruption of microbial membranes may be a central attribute related to the host defense properties of protegrins.

Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease.

Use of planar lipid bilayer membranes for rapid screening of membrane active compounds.

Publisher Summary Planar lipid bilayer membranes (BLMs) have been in use for more than three decades for the study of membrane active compounds) BLMs provide a unique environment that allows the assay of the functional activity of carriers and channels translocating ions across membrane. Although BLMs are in vitro systems that have been used extensively for the measurement of biophysical properties of carriers and channels, their relevance to in vivo phenomena is discussed repeatedly through the more recent technology of patch clamping that has shown that channel properties measured in BLMs correspond qualitatively and often quantitatively to those observed in whole cells. This chapter describes a new BLM perfusion chamber that allows rapid, reversible, and quantitative assay of compounds on the bilayers.

Redistribution of the electric field within the pore contributes to the voltage-dependence of mitochondrial porin channel

The effects of pH on the integral conductance and on the properties of single channels induced by porin from rat liver mitochondria in a lipid bilayer have been studied. When the membrane potential increases, the conductance of the multi-channel membrane decreases more sharply at acidic pH than at neutral or basic pH. The channel is shown to have several states with different conductance and selectivity. The number of levels and their conductance do not depend on pH, while the selectivity as well as the dependence of steady-state probabilities of different levels on the membrane potential are substantially affected by a pH change. This dependence curve steepens in the pH region where charges of carboxyl groups of aspartic and glutamic amino acids are neutralized. It is concluded that at neutral pH the channel gate is controlled by a great number of the positively and negatively charged groups. The high steepness of the conductance-voltage curve in the acidic region suggests that at least 60 positive charges participate in controlling the channel gate. This number, compared with that of the positively charged side chain amino acids per channel, according to the amino acid analysis of the porin, led us to conclude that almost all amino groups of the channel former must pass through the entire membrane potential difference upon random motion of the channel among the states. The assumption that channel closing leads to redistribution of the electric field within the pore, changing the energy of the charges on the voltage sensor, may be the only explanation of this phenomenon.

Channel Formation by a Neurotoxic Beta Amyloid Peptide, Aβ25–35

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder which is accompanied by memory loss and defects in cognitive function. The features in AD brains include extracellular senile plaques, intracellular neurofibrillary tangles, and amyloid proteins deposited in walls of cerebral and meningeal blood vessels. The major component of these pathognomonic lesions of AD has been identified as a 39–43 amino acid long peptide called beta amyloid peptide (Aβ). Aβ is derived from the amyloid β precursor protein (βAPP)1. The thesis that Aβ deposition plays a central role in the pathogenesis of AD has garnered increasing attention. It has been demonstrated that Aβ either is directly neurotoxic to neurons in culture,2,3 or potentiates neuronal vulnerability to excitatory neurotoxins.4,5 Neurotoxic activity has been reported to be located in the fragment of Aβ, Aβ25–35.2 We hypothesize that Aβ and its fragments, such as Aβ25–35, can form ion channels in neuronal membrane, which may allow calcium entry either directly or indirectly, thus leading to neuronal death and neuropathology of AD. In this study, we will characterize the ionic channels formed by Aβ25–35. Preliminary reports of this work have appeared elsewhere.6,7,8