Thermodynamics predicts that transmembrane voltage modulates membrane tension and that this will cause movement. The magnitude and polarity of movement is governed by cell stiffness and surface potentials. Here we confirm these predictions using the atomic force microscope to dynamically follow the movement of voltage-clamped HEK293 cells in different ionic-strength solutions. In normal saline, depolarization caused an outward movement, and at low ionic strength an inward movement. The amplitude was proportional to voltage (about 1 nm per 100 mV) and increased with indentation depth. A simple physical model of the membrane and tip provided an estimate of the external and internal surface charge densities (-5 x 10(-3) C x m(-2) and -18 x 10(-3) C x m(-2), respectively). Salicylate (a negative amphiphile) inhibited electromotility by increasing the external charge density by -15 x 10(-3) C x m(-2). As salicylate blocks electromotility in cochlear outer hair cells at the same concentration, the role of prestin as a motor protein may need to be reassessed.

The cyclotides are a large family of circular mini-proteins containing a cystine knot motif. They are expressed in plants as defense-related proteins, with insecticidal activity. Here we investigate their role in membrane interaction and disruption. Kalata B1, a prototypic cyclotide, was found to induce leakage of the self-quenching fluorophore, carboxyfluorescein, from phospholipid vesicles. Alanine-scanning mutagenesis of kalata B1 showed that residues essential for lytic activity are clustered, forming a bioactive face. Kalata B1 was sequestered at the membrane surface and showed slow dissociation from vesicles. Electrophysiological experiments showed that conductive pores were induced in liposome patches on incubation with kalata B1. The conductance calculated from the current-voltage relationship indicated that the diameter of the pores formed in the bilayer patches is 41-47 Å . Collectively, the findings provide a mechanistic explanation for the diversity of biological functions ascribed to this fascinating family of ultrastable macrocyclic peptides.The cyclotides are a family of topologically unique macrocyclic peptides abundant in plants of the Rubiaceae (coffee) and Violaceae (violet) families. They possess unusual structural and biophysical properties and are composed of a head-to-tail cyclic backbone and a cystine knot (1). The cystine knot is formed by a disulfide bond that penetrates a ring made by two other disulfide bonds and their connecting backbone segments. The structure of kalata B1, the prototypic cyclotide, is illustrated in Fig. 1 (2). The cyclic cystine knot at the core of three-dimensional structure contributes to the exceptional chemical and biological stability of cyclotides (3) and underpins their exciting potential for pharmaceutical and agricultural applications (4).Cyclotides display a diverse range of biological activities, including anti-human immunodeficiency virus (5-8), neurotensin antagonism (9), hemolytic (10), antimicrobial (11), antifouling (12), and pesticidal activities (13-19). Cyclotides have been postulated to be defense-related proteins on the basis of their pesticidal activity and the suite of natural isoforms present in individual plants (20). Little is known about their mechanism of action, but their observed activities potentially might be associated with membrane interactions. Studies utilizing analytical ultracentrifugation (21) have shown that the cyclotide kalata B2 forms specific oligomers in solution, which could potentially have a role in the formation of membrane-spanning pores. A membrane-based mechanism of action is supported by a recent surface plasmon resonance study, which demonstrated that several kalata-like cyclotides bind to phosphatidylethanolamine-containing membranes (22). More recently, the cyclotide cycloviolacin O2 was shown to be cytotoxic to a human lymphoma cell line and induce leakage of calceinloaded HeLa cells (23). NMR studies showed that the binding of kalata B1, and other analogues, to dodecylphosphocholine micelles is modulated by...

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.

It is often assumed that ion channels in cell membrane patches gate independently. However, in the present study nicotinic receptor patch clamp data obtained in cell-attached mode from embryonic chick myotubes suggest that the distribution of steady-state probabilities for conductance multiples arising from concurrent channel openings may not be binomial. In patches where up to four active channels were observed, the probabilities of two or more concurrent openings were greater than expected, suggesting positive cooperativity. For the case of two active channels, we extended the analysis by assuming that 1) individual receptors (not necessarily identical) could be modeled by a five-state (three closed and two open) continuous-time Markov process with equal agonist binding affinity at two recognition sites, and 2) cooperativity between channels could occur through instantaneous changes in specific transition rates in one channel following a change in conductance state of the neighboring channel. This allowed calculation of open and closed sojourn time density functions for either channel conditional on the neighboring channel being open or closed. Simulation studies of two channel systems, with channels being either independent or cooperative, nonidentical or identical, supported the discriminatory power of the optimization algorithm. The experimental results suggested that individual acetylcholine receptors were kinetically identical and that the open state of one channel increased the probability of opening of its neighbor.

Quantitative analysis of patch clamp data is widely based on stochastic models of single-channel kinetics. Membrane patches often contain more than one active channel of a given type, and it is usually assumed that these behave independently in order to interpret the record and infer individual channel properties. However, recent studies suggest there are significant channel interactions in some systems. We examine a model of dependence in a system of two identical channels, each modeled by a continuous-time Markov chain in which specified transition rates are dependent on the conductance state of the other channel, changing instantaneously when the other channel opens or closes. Each channel then has, e.g., a closed time density that is conditional on the other channel being open or closed, these being identical under independence. We relate the two densities by a convolution function that embodies information about, and serves to quantify, dependence in the closed class. Distributions of observable (superposition) sojourn times are given in terms of these conditional densities. The behavior of two channel systems based on two- and three-state Markov models is examined by simulation. Optimized fitting of simulated data using reasonable parameters values and sample size indicates that both positive and negative cooperativity can be distinguished from independence.