Gap junction channels reconstituted in two closely apposed lipid bilayers

Intercellular communication mediated by gap junction channels plays an important role in many cellular processes. In contrast to other channels, gap junction channels span two plasma membranes resulting in an intracellular location for both ends of the junctional pore and the regulatory sites for channel gating. This configuration presents unique challenges for detailed experimental studies of junctional channel physiology and ligand-activation in situ. Availability of an appropriate model system would significantly facilitate future studies of gap junction channel function and structure. Here we show that the double-membrane channel can be reconstituted in pairs of closely apposed lipid bilayers, as experienced in cells. We have trapped the calcium-sensitive dye, arsenazo III (AIII), partially calcium-saturated (AIII-Ca), in one population of connexin32 reconstituted-liposomes, and EGTA in a second one. In such mixtures, the interaction of EGTA with AIII-Ca was measured by a large color shift from blue to red (decreased absorbance at 652 nm). The exchange of these compounds through gap junctions was proportional to these decrements. Results indicate that these connexon-mediated interliposomal channels are functional and are inhibited by the addition of alpha-glycyrrhetinic acid and by flufenamic acid, two gap junction communication inhibitors. Future use of this model system has the potential to improve our understanding of the permeability and modulation of junctional channels in its native intercellular assembly.     

The influence of static magnetic fields on mechanosensitive ion channel activity in artificial liposomes

The influence of static magnetic fields (SMFs) on the activity of recombinant mechanosensitive ion channels (the bacterial mechanosensitive ion channel of large conductance—MscL) following reconstitution into artificial liposomes has been investigated. Preliminary findings suggest that exposure to 80-mT SMFs does not induce spontaneous MscL activation in the absence of mechanical stimulation. However, SMFs do appear to influence the open probability and single channel kinetics of MscL exposed to negative pipette pressure. Typical responses include an overall reduction in channel activity or an increased likelihood of channels becoming trapped open in sub-conducting states following exposure to SMFs. There is a delay in the onset of this effect and it is maintained throughout exposure. Generally, channel activity showed slow or limited recovery following removal of the magnetic field and responses to the magnetic were often reduced or abolished upon subsequent exposures. Pre-exposure of the liposomes to SMFs resulted in reduced sensitivity of MscL to negative pipette pressure, with higher pressures required to activate the channels. Although the mechanisms of this effect are not clear, our initial observations appear to support previous work showing that the effects of SMFs on ion channels may be mediated by changes in membrane properties due to anisotropic diamagnetism of lipid molecules.     

Interplay between Cystic Fibrosis Transmembrane Regulator and Gap Junction Channels Made of Connexins 45, 40, 32 and 50 Expressed in Oocytes

The cystic fibrosis transmembrane regulator (CFTR) is a Cl⁻ channel known to influence other channels, including connexin (Cx) channels. To study the functional interaction between CFTR and gap junction channels, we coexpressed in Xenopus oocytes CFTR and either Cx45, Cx40, Cx32 or Cx50 and monitored junctional conductance (G _j) and its sensitivity to transjunctional voltage (V _j) by the dual voltage-clamp method. Application of forskolin induced a Cl⁻ current; increased G _j approximately 750%, 560%, 64% and 8% in Cx45, Cx40, Cx32 and Cx50, respectively; and decreased sensitivity to V _j gating, monitored by a change in the ratio between G _j steady state and G _j peak (G _jSS/G _jPK) at the pulse. In oocyte pairs expressing just Cx45 in one oocyte (#1) and both Cx45 and CFTR in the other (#2), with negative pulses applied to oocyte #1 forskolin application still increased G _j and decreased the sensitivity to V _j gating, indicating that CFTR activation is effective even when it affects only one of the two hemichannels and that the G _j and V _j changes are not artifacts of decreased membrane resistance in the pulsed oocyte. COOH-terminus truncation reduced the forskolin effect on Cx40 (Cx40TR) but not on Cx32 (Cx32TR) channels. The data suggest a cross-talk between CFTR and a variety of gap junction channels. Cytoskeletal scaffolding proteins and/or other intermediate cytoplasmic proteins are likely to play a role in CFTR-Cx interaction.     

Evaluation and characterization of fetal exposures to low frequency magnetic fields generated by laptop computers

Portable – or “laptop” – computers (LCs) are widely and increasingly used all over the world. Since LCs are often used in tight contact with the body even by pregnant women, fetal exposures to low frequency magnetic fields generated by these units can occur. LC emissions are usually characterized by complex waveforms and are often generated by the main AC power supply (when connected) and by the display power supply sub-system.In the present study, low frequency magnetic field emissions were measured for a set of five models of portable computers. For each of them, the magnetic flux density was characterized in terms not just of field amplitude, but also of the so called “weighted peak” (WP) index, introduced in the 2003 ICNIRP Statement on complex waveforms and confirmed in the 2010 ICNIRP Guidelines for low frequency fields. For the model of LC presenting the higher emission, a deeper analysis was also carried out, using numerical dosimetry techniques to calculate internal quantities (current density and in-situ electric field) with reference to a digital body model of a pregnant woman. Since internal quantities have complex waveforms too, the concept of WP index was extended to them, considering the ICNIRP basic restrictions defined in the 1998 Guidelines for the current density and in the 2010 Guidelines for the in-situ electric field. Induced quantities and WP indexes were computed using an appropriate original formulation of the well known Scalar Potential Finite Difference (SPFD) numerical method for electromagnetic dosimetry in quasi-static conditions.     

Pilot study of extremely low frequency magnetic fields emitted by transformers in dwellings. Social aspects

A large number of epidemiologic studies examining the potential effect of residential exposure to extremely-low frequency (ELF) magnetic fields and childhood leukemia have been published. Two pooled analyses [Ahlbom A, Day N, Feychting M, Roman E, Skinner J, Dockerty J, Linet M, et al. (2000). A pooled analysis of magnetic fields and childhood leukaemia. Br J Cancer. 83(5):692–698; Greenland S, Sheppard AR, Kaune WT, Poole C, Kelsh AM (2000). A pooled analysis of magnetic fields, wire codes, and childhood leukemia. Epidemiology. 11(6):624–634], which included the major epidemiologic studies on ELF magnetic fields and childhood leukemia showed twofold increase in childhood leukemia risk in association with residential ELF exposure above 0.3–0.4 μT. Based on “limited” epidemiologic evidence linking ELF exposure to childhood leukemia and “inadequate evidence” for carcinogenicity of ELF in rodent bioassays, the International Agency for Research on Cancer (IARC) classified ELF magnetic fields as a possible human carcinogen (2B classification) [International Agency for Research on Cancer (IARC) (2002). Non-ionizing radiation, Part 1: Static and extremely low-frequency (ELF) electric and magnetic fields. IARC monographs on the evaluation of carcinogenic risks to humans. Vol. 80. IARC Press: Lyon], confirmed by WHO on the basis of studies published after 2000 [World Health Organization. Extremely low frequency fields. In: 238 Environmental health criteria, Geneva: WHO; 2007]. The analysis of more recent studies of ELF magnetic fields and childhood leukemia had small findings and propose methodological improvements concerning the uncertainties in epidemiological approaches and exposure assessment, bias in selection of controls [Kheifets L, Oksuzyan S (2008). Exposure assessment and other challenges in non-ionizing radiation studies of childhood leukaemia. Radiat Prot Dosimetry. 132(2):139–147]. By the end of 2010, 37 countries had been identified for possible participation in the International study TRANSEXPO. The pilot work has been completed in five countries (Finland, Hungary, Israel, Switzerland and Bulgaria). In 2008, Bulgaria through the National Centre of Public Health Protection joined with pilot study in TRANSEXPO Project. At this first stage of the project our investigation was directed to performing measurements in dwellings with built-in transformer stations, collecting data of population and cancer registry and choosing the epidemiology design feasible for continuing the project. Taking into account the available sources of information in Bulgaria (different registers of the population) needed for epidemiological approach, it was found that the most appropriate epidemiology design would be the nested case-control study. Control group could be collected in accordance with the international requirements for such epidemiological studies. This approach could be modified in the course of the further study in order to ensure achievement of the purposes of the main international requirements of the study.     

The role of voltage-gated Ca2+ channels in neurite growth of cultured chromaffin cells induced by extremely low frequency (ELF) magnetic field stimulation

The ion Ca²⁺ has been shown to play an important role in a wide variety of cellular functions, one of them being related to cell differentiation in which nerve growth factor (NGF) is involved. Chromaffin cells obtained from adrenals of 2- to 3-day-old rats were cultured for 7 days. During this time, these cells were subjected to the application of either NGF or extremely low frequency magnetic fields (ELF MF). Since this induced cell differentiation toward neuronal-like cells, the mechanism by which this occurred was studied. When the L-Ca²⁺ channel blocker nifedipine was applied simultaneously with ELF MF, this differentiation did not take place, but it did when an N-Ca²⁺ channel blocker was used. In contrast, none of the Ca²⁺ channel blockers prevented differentiation in the presence of NGF. In addition, Bay K-8644, an L-Ca²⁺ channel agonist, increased both the percentage of differentiated cells and neurite length in the presence of ELF MF. This effect was much weaker in the presence of NGF. [³H]-noradrenaline release was reduced by nifedipine, suggesting an important role for L-Ca²⁺ channels in neurotransmitter release. Total high voltage Ca²⁺ currents were significantly increased in ELF MF-treated cells with NGF, but these currents in ELF MF-treated cells were more sensitive to nifedipine. Amperometric analysis of catecholamine release revealed that the KCl-induced activity of cells stimulated to differentiate by ELF MF is highly sensitive to L-type Ca²⁺ channel blockers. A possible mechanism to explain the way in which the application of magnetic fields can induce differentation of chromaffin cells into neuronal-like cells is proposed.