Angiotensin II type 2 receptor (AT2 R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons.

BACKGROUND: The angiotensin II (AngII) receptor subtype 2 (AT2 R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration. METHODS: We used immunostaining with characterized antibodies to study the localization of AT2 R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2 R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence. RESULTS: AT2 R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2 R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50 = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1 R antagonist losartan had no effect on capsaicin responses. AT2 R was localized in sensory neurons of human DRG, and nerve fibres in peripheral nerves, skin, urinary bladder and bowel. A majority sub-population (60%) of small-/medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2 R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas. CONCLUSIONS: AT2 R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting.

A novel light source for SICM-SNOM of living cells.

We have developed a novel light source for use in a scanning near-field optical microscope (SNOM or NSOM) based on a nanopipette whose distance from the sample surface is controlled using scanning ion conductance microscopy. The light source is based on the general principle of the chemical reaction between a fluorophore in the pipette and ligand in the bath, to produce a highly fluorescent complex that is continually renewed at the pipette tip. In these experiments we used fluo-3 and calcium, respectively. This complex is then excited with an Ar+ laser, focused on the pipette tip, to produce the light source. This method overcomes the transmission problem of more traditional SNOM probes and has been used to acquire simultaneous high-resolution topographic and optical images of biological samples in physiological buffer. A resolution of approximately 220 nm topographic and approximately 190 nm optical was determined through imaging fixed sea-urchin sperm flagella. Live A6 cells were also imaged, demonstrating the potential of this system for SNOM imaging of living cells.

Simultaneous measurement of Ca2+ and cellular dynamics: combined scanning ion conductance and optical microscopy to study contracting cardiac myocytes.

We have developed a distance modulated protocol for scanning ion conductance microscopy to provide a robust and reliable distance control mechanism for imaging contracting cells. The technique can measure rapid changes in cell height from 10 nm to several micrometers, with millisecond time resolution. This has been demonstrated on the extreme case of a contracting cardiac myocyte. By combining this method with laser confocal microscopy, it was possible to simultaneously measure the nanometric motion of the cardiac myocyte, and the local calcium concentration just under the cell membrane. Despite large cellular movement, simultaneous tracking of the changes in cell height and measurement of the intracellular Ca2+ near the cell surface is possible while retaining the cell functionality.

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.

Hybrid scanning ion conductance and scanning near-field optical microscopy for the study of living cells.

We have developed a hybrid scanning ion conductance and scanning near-field optical microscope for the study of living cells. The technique allows quantitative, high-resolution characterization of the cell surface and the simultaneous recording of topographic and optical images. A particular feature of the method is a reliable mechanism to control the distance between the probe and the sample in physiological buffer. We demonstrate this new method by recording near-field images of living cells (cardiac myocytes).

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.

A conserved tryptophan in pneumolysin is a determinant of the characteristics of channels formed by pneumolysin in cells and planar lipid bilayers.

Pneumolysin is one of the family of thiol-activatable, cytolytic toxins. Within these toxins the amino acid sequence Trp-Glu-Trp-Trp is conserved. Mutations made in this region of pneumolysin, residues 433-436 inclusive, did not affect cell binding or the formation of toxin oligomers in the target cell membrane. However, the mutations did affect haemolysis, leakage of low-molecular-mass metabolites from Lettre cells and the induction of conductance channels across planar lipid bilayers. Of eight modified pneumolysins examined, Trp-433-->Phe showed the smallest amount of haemolysis or leakage (less than 5% of wild type). Pneumolysin-induced leakage from Lettre cells was sensitive to inhibition by bivalent cations but the extent of inhibition varied depending on the modification. Leakage by the mutant Trp-433-->Phe was least sensitive to cation inhibition. The ion-conducting channels formed across planar lipid bilayers exhibit small (less than 30 pS), medium (30 pS-1 nS) and large (more than 1 nS) conductance steps. Small- and medium-sized channels were preferentially closed by bivalent cations. In contrast with wild-type toxin, which formed predominantly small channels, the modified toxin Trp-433-->Phe formed large channels that were insensitive to cation-induced closure. Polysaccharides of molecular mass more than 15 kDa inhibited haemolysis by wild-type toxin, but polysaccharide of up to 40 kDa did not prevent haemolysis by Trp-433-->Phe. Electron microscopy revealed that Trp-433-->Phe formed oligomeric arc and ring structures with dimensions identical with those of wild-type toxin, and that the ratio of arcs to rings formed was the same for wild-type toxin and the Trp-433-->Phe variant. We conclude that the change Trp-433-->Phe affects channel formation at a point subsequent to binding to the cell membrane and the formation of oligomers, and that the size of arc and ring structures revealed by electron microscopy does not reflect the functional state of the channels.

Specialized scanning ion-conductance microscope for imaging of living cells.

A specialized scanning ion conductance microscope (SICM) for imaging living cells has been developed from a conventional patch-clamp apparatus, which uses a glass micropipette as the sensitive probe. In contrast with other types of scanning probe microscope, the SICM probe has significant advantages for imaging living cells: it is most suitable for imaging samples immersed in water solutions; and since the probe senses ion current and does not need physical contact with the sample during the scan, any preliminary preparation of cells (fixation or adherence to a substrate) is unnecessary. We have successfully imaged murine melanocytes in growth medium. The microscope images the highly convoluted surface structures without damaging or deforming them, and reveals the true, three-dimensional relief of the cells. This instrument has considerable ability to operate, potentially simultaneously, in applications as diverse as real-time microscopy, electrophysiology, micromanipulation and drug delivery.

Scanning ion conductance microscopy of living cells.

Currently there is a great interest in using scanning probe microscopy to study living cells. However, in most cases the contact the probe makes with the soft surface of the cell deforms or damages it. Here we report a scanning ion conductance microscope specially developed for imaging living cells. A key feature of the instrument is its scanning algorithm, which maintains the working distance between the probe and the sample such that they do not make direct physical contact with each other. Numerical simulation of the probe/sample interaction, which closely matches the experimental observations, provides the optimum working distance. The microscope scans highly convoluted surface structures without damaging them and reveals the true topography of cell surfaces. The images resemble those produced by scanning electron microscopy, with the significant difference that the cells remain viable and active. The instrument can monitor small-scale dynamics of cell surfaces as well as whole-cell movement.

A novel explanation for fluctuations of ion current through narrow pores.

Fluctuation of ion current, between a high conductance and a low conductance state, through biological ion channels and pores is assumed to arise from conformational changes between an "open" and a "closed" configuration. Here we offer an additional mechanism that arises from changes in ionization of fixed charges within, or at the mouth of, a channel or pore. Our hypothesis, which is based on measurements of ion selectivity alongside ion current, applies to pores through some synthetic membranes and through channels-such as those created by certain toxins-that remain (at least partially) open in the low conductance state. It may also explain the phenomena of "open channel noise" and "substate behavior" that characterize several endogenous ion channels and should be considered when modeling the behavior of such channels.

Pore formation by S. aureus alpha-toxin in liposomes and planar lipid bilayers: effects of nonelectrolytes.

Nonelectrolytes such as polyethylene glycols (PEG) and dextrans (i) promote the association of S. aureus alpha-toxin with liposomes (shown by Coomassie staining) and (ii) enhance the rate and extent of calcein leakage from calcein-loaded liposomes; such leakage is inhibited by H+, Zn2+ and Ca2+ to the same extent as that of nonPEG-treated liposomes. Incubation of liposomes treated with alpha-toxin in the presence of PEG with the hydrophobic photo-affinity probe 3-(trifluoromethyl)-3-m-[125I]iodophenyl) diazirine (125I-TID) labels monomeric and-predominantly-hexameric forms of liposome-associated alpha-toxin; in the absence of PEG little labeling is apparent. At high concentrations of H+ and Zn2+ but not of Ca(2+)-all of which inhibit calcein leakage-the distribution of label between hexamer and monomer is perturbed in favor of the latter. In alpha-toxin-treated planar lipid bilayers from which excess toxin has been washed away, PEGs and dextrans strongly promote the appearance of ion-conducting pores. The properties of such pores are similar in most regards to pores induced in the absence of nonelectrolytes; the differ only in being more sensitive to "closure" by voltage (as are pores induced in cells). In both systems, the stimulation by nonelectrolytes increase with concentration and with molecular mass up to a maximum around 2,000 Da. We conclude (i) that most of the alpha toxin that becomes associated with liposome or planar lipid bilayers does not form active pores and (ii) that the properties of alpha-toxin-induced pores in lipid bilayers can be modulated to resemble those in cells.

Low conductance states of a single ion channel are not 'closed'.

We have used a polymer-exclusion method to estimate the sizes of the high- and low-conductance states of Staphylococcus aureus alpha-toxin channels across planar lipid bilayers. Despite a > 10-fold difference in conductance between high- and low-conductance states, the size differs by < 2-fold. We conclude that factors other than the dimensions have a strong influence on the conductance of alpha-toxin channels. We also show that the high conductance state is destabilized by the presence of high molecular weight polymers outside the channel, compatible with the removal of channel water as the high conductance state "shrinks" to the low conductance state.

Staphylococcus aureus alpha-toxin-induced pores: channel-like behavior in lipid bilayers and patch clamped cells.

The conductance of pores induced by Staphylococcus aureus alpha-toxin in Lettre cells has been compared to that in bilayers composed of synthetic lipids or Lettre cell membrane constituents. Previously described characteristics of toxin-induced conductance changes in lipid bilayers, namely rectification, voltage-dependent closure, and closure at low pH or in the presence of divalent cations (Menestrina, 1986) are displayed also in bilayers prepared from Lettre cell membranes and in patch clamped Lettre cells. It is concluded that endogenous proteins do not affect the properties of alpha-toxin-induced channels significantly and that the relative lack of ion channels in Lettre cells makes them ideal for studies of pore-forming toxins by the patch clamp technique.

Rapid switching of ion current in narrow pores: implications for biological ion channels.

Ions flowing through purely synthetic filters made of polyethylene terephthalate which have been etched to produce narrow pores show: (i) rapid transitions between a high-conducting and a low-conducting state; (ii) selectivity of ion flow; and (iii) inhibition by divalent cations and protons. These features resemble those displayed by many biological ion channels. We interpret our results in terms of the special properties of ion conductance at an interface that may be observed whenever the contribution of bulk conductance is minimal.

Membrane damage: common mechanisms of induction and prevention.

Common features in the induction of pores by various agents are as follows: induction is stochastic and progressive; damage by different agents is often synergistic and limited. The prevention of membrane damage is affected by trivalent and divalent cations, by low pH, by low ionic strength and by high osmotic pressure. The inhibitory role of protons and divalent cations is considered in greater detail: pore-forming agents can be classified into two groups: channels across planar lipid bilayers induced by the first group display voltage-sensitive, reversible inhibition by divalent cations; channels of the second group show voltage-insensitive, irreversible inhibition by divalent cations. A search for the ligands to which divalent cations and protons bind has proved elusive. Comparison with the phenomenon of 'surface conductance' through narrow apertures, that is manifest in the absence of any pore-forming agent, may prove fruitful.

Sterol specific inactivation of gramicidin A induced membrane cation permeability.

Channel inactivation, a time-dependent decrease of the high-cationic permeability induced by gramicidin A, has been found both in cholesterol containing red blood cell membranes and lipid bilayers (Schagina et al., (1989) Biochim. Biophys. Acta 978, 145-150). The rate of channel inactivation strongly depends on the phospholipid to cholesterol molar ratio of the membrane. The channel inactivation is suggested to be the result of an interaction between gramicidin and cholesterol in a stoichiometry of 1:5. Cholesterol dependent inactivation is shown also for gramicidin A analogs: tryptophan-N-formylated gramicidin A, o-pyromellitilgramicidin and malonylbisdesformylgramicidin. When cholesterol in the membrane is substituted by sitosterol, the inactivation of gramicidin-induced cation permeability is preserved, while in the presence of either ergosterol or 7-dehydrocholesterol no indication of the channel inactivation is observed. Thus, the structure of the 'B', ring, not the apolar tail of the sterol molecule, appears to be important in the inactivation process.

Differential sensitivity of pneumolysin-induced channels to gating by divalent cations.

The induction of channels across planar lipid bilayers by purified, recombinant pneumolysin (a hemolytic protein from Streptococcus pneumoniae) has been studied by measuring increases in electrical conductivity. Pneumolysin-induced channels exhibit a wide range of single channel conductances (less than 50 pS to greater than 1 nS at 0.1 M KCl). Channels can be categorized on the basis of their K+:Cl- selectivity: the smallest channels are strongly cation selective, with t+ (the cation transference number) approaching 1.0; the largest channels are unselective (t+ approximately 0.5). Channels tend to remain open at all voltages (-150 to 150 mV); only the smallest channels exhibit any rectification. In the presence of divalent cations (1-5 mM Zn2+; 10-20 mM Ca2+), small (less than 50 pS) and medium-sized (50 pS to 1 nS) channels are closed in a voltage-dependent manner (more closure at higher voltages); at 0 voltage channels reopen. Overall selectivity is reduced by divalent cations, compatible with small, selective channels being closed preferentially to large, nonselective ones. It is concluded that a single molecular species (pneumolysin) induces multiple-sized channels that can be categorized by cation:anion selectivity and by their sensitivity to closure by divalent cations.

Cholesterol-dependent gramicidin A channel inactivation in red blood cell membranes and lipid bilayer membranes.

The exchange diffusions of tracer cations (22Na+, 86Rb+) are studied on gramicidin-A-treated red blood cell (RBC) membranes. A time-dependent decrease in cation permeability has been observed and has been considered to be the result of a channel inactivation process. The channel inactivation appears at 20 and 30 degrees C but not at a temperature as low as 6 degrees C. The gramicidin A channel inactivation can be monitored by a conductivity decay of molecular lipid membranes (BLM) prepared either from cholesterol or from a mixture of cholesterol and phospholipids but not of pure phosphatidylethanolamine. The role of cholesterol in the channel inactivation is discussed.