Igor Vodyanoy

Functional localization of single active ion channels on the surface of a living cell.

The spatial distribution of ion channels in the cell plasma membrane has an important role in governing regional specialization, providing a precise and localized control over cell function. We report here a novel technique based on scanning ion conductance microscopy that allows, for the first time, mapping of single active ion channels in intact cell plasma membranes. We have mapped the distribution of ATP-regulated K+ channels (KATP channels) in cardiac myocytes. The channels are organized in small groups and anchored in the Z-grooves of the sarcolemma. The distinct pattern of distribution of these channels may have important functional implications.

Cell volume measurement using scanning ion conductance microscopy.

We report a novel scanning ion conductance microscopy (SICM) technique for assessing the volume of living cells, which allows quantitative, high-resolution characterization of dynamic changes in cell volume while retaining the cell functionality. The technique can measure a wide range of volumes from 10(-19) to 10(-9) liter. The cell volume, as well as the volume of small cellular structures such as lamelopodia, dendrites, processes, or microvilli, can be measured with the 2.5 x 10(-20) liter resolution. The sample does not require any preliminary preparation before cell volume measurement. Both cell volume and surface characteristics can be simultaneously and continuously assessed during relatively long experiments. The SICM method can also be used for rapid estimation of the changes in cell volume. These are important when monitoring the cell responses to different physiological stimuli.

Dynamic assembly of surface structures in living cells

Although the dynamics of cell membranes and associated structures is vital for cell function, little is known due to lack of suitable methods. We found, using scanning ion conductance microscopy, that microvilli, membrane projections supported by internal actin bundles, undergo a life cycle: fast height-dependent growth, relatively short steady state, and slow height-independent retraction. The microvilli can aggregate into relatively stable structures where the steady state is extended. We suggest that the intrinsic dynamics of microvilli, combined with their ability to make stable structures, allows them to act as elementary “building blocks” for the assembly of specialized structures on the cell surface.

Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system.

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.

High-resolution scanning patch-clamp: new insights into cell function

Cell specialization is often governed by the spatial distribution of ion channels and receptors on the cell surface. So far, little is known about functional ion channel localization. This is due to a lack of satisfactory methods for investigating ion channels in an intact cell and simultaneously determining the channels‘ positions accurately. We have developed a novel high‐resolution scanning patch‐clamp technique that enables the study of ion channels, not only in small cells, such as sperm, but in submicrometer cellular structures, such as epithelial microvilli, fine neuronal dendrites, and, particularly, T‐tubule openings of cardiac myocytes. In cardiac myocytes, as in most excitable cells, action potential propagation depends essentially on the properties of ion channels that are functionally and spatially coupled. We found that the L‐type calcium and chloride channels are distributed and colocalized in the region of T‐tubule openings, but not in other regions of the myocyte. In addition, chloride channels were found in narrowly defined regions of Z‐grooves. This finding suggests a new synergism between these types of channels that may be relevant for action potential propagation along the T‐tubule system and excitation‐contraction coupling.

The use of scanning ion conductance microscopy to image A6 cells

BACKGROUND Continuous high spatial resolution observations of living A6 cells would greatly aid the elucidation of the relationship between structure and function and facilitate the study of major physiological processes such as the mechanism of action of aldosterone. Unfortunately, observing the micro-structural and functional changes in the membrane of living cells is still a formidable challenge for a microscopist. METHOD Scanning ion conductance microscopy (SICM), which uses a glass nanopipette as a sensitive probe, has been shown to be suitable for imaging non-conducting surfaces bathed in electrolytes. A specialized version of this microscopy has been developed by our group and has been applied to image live cells at high-resolution for the first time. This method can also be used in conjunction with patch clamping to study both anatomy and function and identify ion channels in single cells. RESULTS This new microscopy provides high-resolution images of living renal cells which are comparable with those obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Continuous 24h observations under normal physiological conditions showed how A6 kidney epithelial cells changed their height, volume, and reshaped their borders. The changes in cell area correlated with the density of microvilli on the surface. Surface microvilli density ranged from 0.5 microm(-2) for extended cells to 2.5 microm(2) for shrunk cells. Patch clamping of individual cells enabled anatomy and function to be correlated. CONCLUSIONS Scanning ion conductance microscopy provides unique information about living cells that helps to understand cellular function. It has the potential to become a powerful tool for research on living renal cells.

Lipid packing stress and polypeptide aggregation: alamethicin channel probed by proton titration of lipid charge

Lipid membranes are not passive, neutral scaffolds to hold membrane proteins. In order to examine the influence of lipid packing energetics on ion channel expression, we study the relative probabilities of alamethicin channel formation in dioleoylphosphatidylserine (DOPS) bilayers as a function of pH. The rationale for this strategy is our earlier finding that the higher-conductance states, corresponding to larger polypeptide aggregates, are more likely to occur in the presence of lipids prone to hexagonal HII-phase formation (specifically DOPE), than in the presence of lamellar L alpha-forming lipids (DOPC). In low ionic strength NaCl solutions at neutral pH, the open channel in DOPS membranes spends most of its time in states of lower conductance and resembles alamethicin channels in DOPC; at lower pH, where the lipid polar groups are neutralized, the channel probability distribution resembles that in DOPE. X-Ray diffraction studies on DOPS show a progressive decrease in the intrinsic curvature of the constituent monolayers as well as a decreased probability of HII-phase formation when the charged lipid fraction is increased. We explore how proton titration of DOPS affects lipid packing energetics, and how these energetics couple titration to channel formation.

Esmolol is antiarrhythmic in doxorubicin‐induced arrhythmia in cultured cardiomyocytes – determination by novel rapid cardiomyocyte assay

Cardiac toxicity is an uncommon but potentially serious complication of cancer therapy, especially with anthracyclines. One of the most effective anticancer drugs is doxorubicin, but its value is limited by the risk of developing cardiomyopathy and ventricular arrhythmia. When applied to a network of periodically contracting cardiomyocytes in culture, doxorubicin induces rhythm disturbances. Using a novel rapid assay based on non‐invasive ion‐conductance microscopy we show that the β‐antagonist esmolol can restore rhythm in doxorubicin‐treated cultures of cardiomyocytes. Moreover, esmolol pre‐treatment can protect the culture from doxorubicin‐induced arrhythmia.

Experimental Comparative Study of the Applicability of Infrared Techniques for Non-destructive Evaluation of Paintings

Abstract Noninvasive methods of near-infrared, short-wave infrared, and thermographic inspection of artwork are described in this article and compared in terms of their ability to reveal both hidden graphite underdrawings and subsurface degradations. This inspection aids the understanding of the artists work methods and locates hidden areas of damage. While all three inspection methods are suitable for locating sketches and changes in composition, this study has proven that thermographic methods are very useful in detecting structural defects such as delaminations and cavities, as demonstrated with experiments conducted on test samples and real paintings.

Stochastic resonance at molecular level: The Poisson wave model

Noise-facilitated signal transduction or the ‘stochastic resonance’ (SR) phenomenon, originally proposed as an explanation of the periodic recurrences of the Earth’s ice ages [1], has been attracting rapidly growing interest from researchers working in different areas of science from physics to biology [2,3]. Only a few years ago it was generally accepted that SR can occur only in dynamical systems subjected to random forcing. Later it was hypothesized [4], and then shown both experimentally and theoretically [5,6], that the simplest ‘stochastic resonator’ consists only of signal, noise, and a threshold device. We introduce yet another class of systems where a noise-induced increase in the output signal-to-noise ratio can be observed [7]. These systems are both non-dynamical and threshold free. We find SR in a very general model—a random pulse train where the probability of pulse generation is exponentially dependent on an input which is composed of a sine-wave signal plus random noise. We demonstrate tha...