The use of electrophysiological and molecular biology techniques has shed light on reactive oxygen species (ROS)-induced impairment of surface and internal membranes that control cellular signaling. These deleterious effects of ROS are due to their interaction with various ion transport proteins underlying the transmembrane signal transduction, namely, 1) ion channels, such as Ca2+ channels (including voltage-sensitive L-type Ca2+currents, dihydropyridine receptor voltage sensors, ryanodine receptor Ca2+-release channels, andd- myo-inositol 1,4,5-trisphosphate receptor Ca2+-release channels), K+ channels (such as Ca2+-activated K+ channels, inward and outward K+ currents, and ATP-sensitive K+ channels), Na+ channels, and Cl− channels; 2) ion pumps, such as sarcoplasmic reticulum and sarcolemmal Ca2+pumps, Na+-K+-ATPase (Na+ pump), and H+-ATPase (H+ pump); 3) ion exchangers such as the Na+/Ca2+exchanger and Na+/H+exchanger; and 4) ion cotransporters such as K+-Cl−, Na+-K+-Cl−, and Pi-Na+cotransporters. The mechanism of ROS-induced modifications in ion transport pathways involves 1) oxidation of sulfhydryl groups located on the ion transport proteins, 2) peroxidation of membrane phospholipids, and 3) inhibition of membrane-bound regulatory enzymes and modification of the oxidative phosphorylation and ATP levels. Alterations in the ion transport mechanisms lead to changes in a second messenger system, primarily Ca2+ homeostasis, which further augment the abnormal electrical activity and distortion of signal transduction, causing cell dysfunction, which underlies pathological conditions.

Cytotoxic peptides are relatively small cationic molecules such as those found 1) in venoms, e.g., melittin in bee, scorpion toxins in scorpion, pilosulin 1 in jumper ant, and lycotoxin I and II in wolf spider; 2) in skin secretions (e.g., magainin I and II from Xenopus laevis, dermaseptin from frog, antimicrobials from carp) and cells of the immune system (e.g., insect, scorpion, and mammalian defensins and cryptdins); 3) as autocytotoxicity peptides, e.g., amylin cytotoxic to pancreatic beta-cells, prion peptide fragment 106-126 [PrP-(106-126)], and amyloid beta-protein (AbetaP) cytotoxic to neurons; and 4) as designed synthetic peptides based on the sequences and properties of naturally occurring cytotoxic peptides. The small cytotoxic peptides are composed of beta-sheets, e.g., mammalian defensins, AbetaP, amylin, and PrP-(106-126), whereas the larger cytotoxic peptides have several domains composed of both alpha-helices and beta-sheets stabilized by cysteine bonds, e.g., scorpion toxins, scorpion, and insect defensins. Electrophysiological and molecular biology techniques indicate that these structures modify cell membranes via 1) interaction with intrinsic ion transport proteins and/or 2) formation of ion channels. These two nonexclusive mechanisms of action lead to changes in second messenger systems that further augment the abnormal electrical activity and distortion of the signal transduction causing cell death.

SUMMARY1. Protein-membrane interaction includes the interaction of proteins with intrinsic receptors and ion transport pathways and with membrane lipids. Several hypothetical interaction models have been reported for peptide-induced membrane destabilization, including hydrophobic clustering, electrostatic interaction, electrostatic followed by hydrophobic interaction, wedge ؋ type incorporation and hydrophobic mismatch.2. The present review focuses on the hypothesis of protein interaction with lipid membranes of those unchaperoned positively charged and misfolded proteins that have hydrophobic regions. We advance the hypothesis that protein misfolding that leads to the exposure of hydrophobic regions of proteins renders them potentially cytotoxic. Such proteins include prion, amyloid ␤ protein (A ␤ P), amylin, calcitonin, serum amyloid and C-type natriuretic peptides. These proteins have the ability to interact with lipid membranes, thereby inducing membrane damage and cell malfunction.3. We propose that the most significant mechanism of membrane damage induced by hydrophobic misfolded proteins is mediated via the formation of ion channels. The hydrophobicity based toxicity of several proteins linked to neurodegenerative pathologies is similar to those observed for antibacterial toxins and viral proteins. 4. It is hypothesized that the membrane damage induced by amyloids, antibacterial toxins and viral proteins represents a common mechanism for cell malfunction, which underlies the associated pathologies and cytotoxicity of such proteins.

The annexins are water soluble proteins possessing a hydrophilic surface, which belong to a family of proteins which (a) bind ('annex') both calcium and phospholipids, and (b) form voltage-dependent calcium channels within planar lipid bilayers. Annexins types are diverse (94 annexins in 45 species) and they belong to an enormous multigene family that ranges throughout all eukaryotic kingdoms. Although the structure of these proteins is now well known their functional and physiological roles remain largely unknown and circumstantial. Various experimental approaches provided evidence that annexins function as Ca(2+) channels that could act as regulators of membrane fusion. The identity of annexins is derived from the conserved 34 kDa C-terminal domain which comprises four repeats - except for annexin VI, with eight repeats - of a sequence of approximately seventy amino acids, which holds the area known as the 'endonexin fold', with its identifying GXGTDE. Annexins have been placed into three subgroups of (1) tetrad core and short amino terminal, (2) tetrad core and long amino terminal, and (3) octad core and short amino terminal. The repeats are highly conserved, each forming a compact alpha-helical domain comprising five alpha-helices wound in a right-handed superhelix. Four domains are formed, arranged in a nearly flat and cyclical array, with domains I and IV, and II and III respectively forming two tightly organised modules with almost twofold symmetry. A hydrophilic pore lies at the centre of the molecule, forming a prominent ion channel coated with charged and highly conserved residues. The annexin molecule is slightly curved, with both a convex and a concave face. The cation/anion permeability ratios and the selectivity sequence of the ion channels formed by several annexins confirm the selectivity of the annexins for Ca(2+) over other divalent cations, and reveals the importance of structural sites, e.g. amino acid positions 17, 78, 95 and 112 for the identification of the ion channel's position, function and regulation. Some are sensitive to low doses of the phenothiazine drugs, trifluoperazine (an anti-schizophrenia drug) and promethazine (anti nausea drug) La(3+) and Cd(2+), (blockers of voltage-gated Ca(2+) channels) nifedipine (an inhibitor of non-activating Ca(2+) channels). There are two main competing models used to explain in vitro ion channel activity of annexins: one involves changes in the conductance of ion via electrostatic disturbance of the membrane surface; the other involves a much more extensive alteration in protein structure and a correspondingly deeper penetration into the membrane.

A comparison is made of two types of chloride-selective channel in skeletal muscle sarcoplasmic reticulum (SR) vesicles incorporated into lipid bilayers. The I/V relationships of both channels, in 250/50 mM Cl- (cis/trans), were linear between -20 and +60 mV (cis potential,) reversed near Ecl and had slope conductances of approximately 250 pS for the big chloride (BCl) channel and approximately 70 pS for the novel, small chloride (SCl) channel. The protein composition of vesicles indicated that both channels originated from longitudinal SR and terminal cisternae. BCl and SCl channels responded differently to cis SO4(2-) (30-70 mM), 4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid (8-80 microM) and to bilayer potential. The BCl channel open probability was high at all potentials, whereas SCl channels exhibited time-dependent activation and inactivation at negative potentials and deactivation at positive potentials. The duration and frequency of SCl channel openings were minimal at positive potentials and maximal at -40 mV, and were stationary during periods of activity. A substate analysis was performed using the Hidden Markov Model (S. H. Chung, J. B. Moore, L. Xia, L. S. Premkumar, and P. W. Gage, 1990, Phil. Trans. R. Soc. Lond. B., 329:265-285) and the algorithm EVPROC (evaluated here). SCl channels exhibited transitions between 5 and 7 conductance levels. BCl channels had 7-13 predominant levels plus many more short-lived substates. SCl channels have not been described in previous reports of Cl- channels in skeletal muscle SR.