Hans-Albert Kolb

Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells

SummaryFor patch-clamp measurements cultured kidney (OK) cells were exposed to osmotic and mechanical stress. Superfusion of a cell in whole cell configuration with hypotonic media (190 mOsm) evokes strong depolarization, which is reversible by returning to the isotonic bath medium. In the cell-attached configuration the exposure to hypotonic media evokes up to six ion channels of homogeneous single-channel properties in the membrane patch. Subsequently, the channels became activated after a time lag of a few seconds. At an applied membrane potential of 0 mV, the corresponding membrane current is directed inward and shows a transient behavior in the time range of minutes. In the same membrane patch these ion channels can be activated by application of negative hydrostatic pressure. The channel has a single-channel conductance of about 22 pS and is permeable to Na+ and K+ as well as to Cl−. It is suggested that volume regulation involves mechanoreceptor-operated ion channels.

Analysis of the multi-pore system of alamethicin in a lipid membrane

SummaryThe electrical properties of an alamethicin multi-pore system have been studied by voltage-jump current-relaxation experiments (this paper) and by autocorrelation and spectral analysis (following paper). With these methods a slow time constant and a fast time constant were observed which differ by about one to three orders of magnitude depending on the experimental conditions. Steady-state current and time constants were analyzed as functions of voltage, alamethicin concentration and temperature. Within experimental error the data obtained with these different methods are in good agreement. The experimentally measured relation between the voltage and alamethicin concentration dependence of the slow relaxation time fits into a model of an alamethicin pore which adopts consecutive pore states and which decays only from the lowest state. It indicates that the uptake of one alamethicin molecule by the existing pore and, in formal equivalence, the transfer of about one positive elementary charge across the membrane are associated with the transition from a given pore conductance state to the next higher state. From the voltage and alamethicin concentration dependence of the pore formation rate evidence shows that a hexameric preaggregate exists at the membrane interface out of which two to three molecules are simultaneously inserted into the membrane to form the pore nucleus. The effects of different voltage pretreatment on the experimentally determined parameters have been investigated and are discussed in detail.

Influence of membrane thickness and ion concentration on the properties of the gramicidin A channel Autocorrelation, spectral power density, relaxation and single-channel studies

The properties of the gramicidin A channel in membranes made from a series of monoglycerides have been studied. In agreement with previous studies, the dissociation rate constant kD of the dimeric channel was found to increase strongly with increasing chain length of the monoglyceride, corresponding to a decrease of the mean life-time of the channel. The value of kD, however, was not strictly correlated with the membrane thickness, as seen from a comparison of membranes with different solvent content. Furthermore, the life-time of the channel increased with the concentration of the permeable ion. This effect was tentatively explained by an electrostatic stabilization of the channel. The single-channel conductance lambda was found to decrease with increasing membrane thickness d, if d was varied by increasing the chain length of the lipid. On the other hand, if d was changed by varying the solvent content of the membranes formed from one and the same lipid, lambda remained constant. These observations were explained by the assumption of local inhomogeneities in the membrane thickness. A striking difference between the lambda values obtained from autocorrelation analysis in the presence of many presence of many channels (lambda a) and those obtained from single-channel experiments (lambda sc) occurred with membranes from longer chain-length monoglycerides. This difference disappeared at low ion concentrations. Electrostatic interactions between channels in local clusters were proposed for an interpretation of these findings.

Identification of a voltage-dependent anion channel in the apical membrane of a Cl−-secretory epithelium (MDCK)

An anion-selective channel of large unit conductance is present in apical membranes of a secretory epithelial cell. For cell-attached patch-clamp configuration on cultured MDCK cells channels with a conductance of about 460 pS are observed at 37° C. The channel becomes spontaneously activated only in rare cases. In inside-out membrane patches the probability of observing this type of channel increased significantly suggesting that the channel is controlled by an up to now unknown mechanism. The channel shows a voltage-dependent burst kinetic.

Correlation analysis of electrical noise in lipid bilayer membranes: kinetics of gramicidin A channels.

SummaryIf a membrane contains ion-conducting channels which form and disappear in a random fashion, an electric current which is passed through the membrane under constant voltage shows statistical fluctuations. Information on the kinetics of channel formation and on the conductance of the single channel may be obtained by analyzing the electrical noise generated in a membrane containing a great number of channels. For this purpose the autocorrelation function of the current noise is measured at different concentrations of the channel-forming substance. As a test system for the application of this technique we have used lipid bilayer membranes doped with gramicidin A. From the correlation time of the current noise generated by the membrane, the rate constants of formation (kR) and dissociation (kD) of the channels could be determined. In addition, the mean square of the current fluctuations yielded the single-channel conductance Λ. The values ofkR,kD, and Λ obtained from the noise analysis agreed closely with the values determined by relaxation measurments and single-channel experiments.

Potassium Channels in Excitable and Non-excitable Cells

Potassium channels play a crucial role in determining the resting membrane potential, time course, amplitude and polarity of electrical changes in most types of cells. At rest the membrane potential of a typical cell is positive to the potassium equilibrium potential (E K). When K+ channels are activated and open, the cell hyperpolarizes and the opening probability of depolarization-dependent calcium and sodium channels is reduced.

Three-dimensional cell growth on structures fabricated from ORMOCER® by two-photon polymerization technique

Two-photon polymerization technique was applied to generate three-dimensional (3D) scaffold-like structures using the photosensitive organic—inorganic hybrid polymer ORMOCER®. The structures were studied with respect to potential applications as scaffold for tissue engineering. Cell counting and comet assay, respectively, demonstrated that doubling time and DNA strand breaks of CHO cells, GFSHR-17 granulosa cells, GM-7373 endothelial cells, and SH-SY5Y neuroblastoma cells were not affected by ORMOCER®. ORMOCER® related alteration of formation of tissue specific cell-to-cell adhesions like gap junctions was ruled out by double whole-cell patch-clamp technique. Additionally, growth of cells on the vertical surfaces of 3D structures composed of ORMOCER® is shown.

Hypotonic shock evokes opening of Ca2+-activated K channels in opossum kidney cells.

Using the patch clamp technique we show that exposure of opossum kidney cells to hypotonic shock evokes an outward rectifying potassium current. The corresponding single channel slope conductance approaches 15 pS at depolarizing voltages. The K current also becomes activated after addition of the ionophore A23187 to an isotonic bath medium containing Ca2+. We therefore conclude that the K selective channels are modulated by an elevation of cytoplasmic Ca2+. Evidence is presented that release of Ca2+ from internal stores is involved.

Cytoskeleton and ion movements during volume regulation in cultured PC12 cells

SummaryThe present study investigates the role of cytoskeletal elements, microtubules and microfilaments, on ion transport systems activated during volume regulatory processes in PC12 pheochromocytoma cells. Disruption of microtubule network by colchicine (0.1 mm) or vinblastine sulfate (10 μm) has no significant effect on PC12 cell hydration or on changes of the intracellular K+, Cl− and Na+ content observed in hypo-osmotic conditions. Disruption of microfilament network by cytochalasin B strongly affects volume regulation in a dose-dependent manner. Cytochalasin B leads to a potentiation of the initial cell swelling and the regulatory volume decrease is suppressed. Although, the internal K+ and Cl− level decreases significantly, as demonstrated by measurements of intracellular ion content and 86Rb fluxes. Using the patch-clamp technique, we could demonstrate in PC12 cell membranes an ion channel whose gating is affected by application of a negative hydrostatic pressure (mechanical stress) to the membrane patch, by exposure of the cell to hypoosmotic medium (osmotic stress), or by disruption of the microfilament network with cytochalasin B.

Possible mechanism of entrapment of neuraminidase-treated lymphocytes in the liver.

Neuraminidase-treated rat lymphocytes adhere strongly to rat hepatocytes in vitro. Binding between cells is due to stereo-specific interactions between a mammalian hepatic membrane lectin and galactosyl residues which are exposed on the lymphocyte surface after removal of sialic acid residues. The hepatic galactose specific lectin may play a role in the trapping of recirculating desialylated lymphocytes in the liver.