Electromagnetic fields (UHF) increase voltage sensitivity of membrane ion channels; possible indication of cell phone effect on living cells.

The effects of ultra high frequency (UHF) nonionizing electromagnetic fields (EMF) on the channel activities of nanopore forming protein, OmpF porin, were investigated. The voltage clamp technique was used to study the single channel activity of the pore in an artificial bilayer in the presence and absence of the electromagnetic fields at 910 to 990 MHz in real time. Channel activity patterns were used to address the effect of EMF on the dynamic, arrangement and dielectric properties of water molecules, as well as on the hydration state and arrangements of side chains lining the channel barrel. Based on the varied voltage sensitivity of the channel at different temperatures in the presence and absence of EMF, the amount of energy transferred to nano-environments of accessible groups was estimated to address the possible thermal effects of EMF. Our results show that the effects of EMF on channel activities are frequency dependent, with a maximum effect at 930 MHz. The frequency of channel gating and the voltage sensitivity is increased when the channel is exposed to EMF, while its conductance remains unchanged at all frequencies applied. We have not identified any changes in the capacitance and permeability of membrane in the presence of EMF. The effect of the EMF irradiated by cell phones is measured by Specific Absorption Rate (SAR) in artificial model of human head, Phantom. Thus, current approach applied to biological molecules and electrolytes might be considered as complement to evaluate safety of irradiating sources on biological matter at molecular level.

Electromagnetic field (EMF) effects on channel activity of nanopore OmpF protein.

In this study, the effects of nonionizing electromagnetic fields (EMF; 925 MHz) on the OmpF porin channel have been characterized at the single-channel level. Channel activity was recorded in real time by the voltage clamp method. Our results showed an increase in the frequency of channel gating and voltage sensitivity. The effects of EMF lasted for several milliseconds after the field source was terminated. However, the conductance levels of channels did not change significantly. Thermal effects of EMF on single-channel properties are a possible cause, based on theoretical evaluation of results that were comparable to those seen in conventional experiments at different temperatures. We conclude that EMF affects both the dynamics and conformation of the channel, either directly by affecting critical amino acid side-chain arrangement, or indirectly, via the electrolyte or the lipid membrane.

Effects of novel antituberculosis agents on OmpF channel activity.

Nanopore forming proteins spanning the outer membrane mediate in the diffusion of hydrophilic chemicals through the hydrophobic bacterial cell wall. In this study, the effects of two novel anti-TB derivatives, ethyl alpha-[5-(5-nitro-2-thienyl)-1,3,4-thiadiazole-2-ylthio] acetates and propyl alpha-[5-(5-nitro-2-thienyl)-1,3,4-thiadiazole-2-ylthio] acetates, on OmpF channel reconstituted in artificial bilayers were evaluated by voltage clamp technique. Surprisingly, ethyl derivative (MIC > or = 6.75 microg/ml) showed no effects on OmpF channel activity but the propyl derivative (MIC=0.39 microg/ml) reduced the channel conductance considerably and changed the gating pattern of the channel. The findings obtained here at molecular level, might shed light on better understanding of the actual mechanism(s) by which the novel anti-TB agents permeate through the cell wall of the Mycobacterium tuberculosis.

Voltage-gating of Escherichia coli porin: a cystine-scanning mutagenesis study of loop 3.

Porins, such as Escherichia coli OmpF, provide the only reported example of a voltage-gated channel where the three-dimensional structure is known to high resolution. Mutations that affect voltage-gating are clustered around the eyelet region, which is a mid-channel constriction caused by a polypeptide loop (L3) folding inside the lumen of this beta-barrel pore. These data, combined with molecular dynamics simulations, indicate that voltage-gating may involve L3 displacement. We have constructed six double cysteine OmpF mutants, five of which form disulphide bonds fixing L3 in the conformation determined by X-ray crystallography. These channels have altered single-channel conductances but unimpaired voltage-gating. The data show that L3 movement is not required for voltage-gating.

Altered voltage sensitivity of mutant OmpC porin channels.

Single OmpC porin channels have been reconstituted in planar bilayer membranes. Wild-type OmpC forms trimers which are largely insensitive to voltages below 250 mV.A single-point mutation of the ompC gene has been prepared resulting in replacement of Trp56 by Cys in the eyelet region of the channel wall in a highly conserved segment of the polypeptide. The monomeric channels of which the trimer is composed have smaller conductivity in 1 M NaCl (400 +/- 20 pS, mean and S.E.M., n=30) and increased voltage sensitivity by comparison with the wild-type under similar conditions, whereas other (Dex) mutants form larger channels and display different behaviour. Further, by treatment in SDS solutions at different temperatures, the W56C mutant has been shown to be less stable than either the wild-type or the Dex mutants.

Brucella Omp2a and Omp2b porins: single channel measurements and topology prediction.

Brucella usually carry two highly homologous genes (omp2a and omp2b) for porin-like proteins. In several B. abortus biovars the omp2a gene has a large deletion compared to other Brucella omp2's. In this study we have measured Omp2 pore activity in planar bilayers. Omp2b exhibits well-defined trimeric channel activity whilst Omp2a forms monomeric pores of variable size which are smaller than Omp2b. No sequence homology exists between Omp2 and porins of known structure, so hydrophobic moment analysis has been used to model their membrane topology. From this it appears likely that the deletion removes the crucial L3 internal loop.

Gating and conduction of nano-channel forming proteins: a computational approach.

Monitoring conformational changes in ion channels is essential to understand their gating mechanism. Here, we explore the structural dynamics of four outer membrane proteins with different structures and functions in the slowest nonzero modes of vibration. Normal mode analysis was performed on the modified elastic network model of channel in the membrane. According to our results, when membrane proteins were analyzed in the dominant mode, the composed pores, TolC and α-hemolysin showed large motions at the intramembrane ß-barrel region while, in other porins, OmpA and OmpF, largest motions observed in the region of external flexible loops. A criterion based on equipartition theorem was used to measure the possible amplitude of vibration in channel forming proteins. The current approach complements theoretical and experimental techniques including HOLE, Molecular Dynamics (MD), and voltage clamp used to address the channel's structure and dynamics and provides the means to conduct a theoretical simultaneous study of the structure and function of the channel. An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:3.

Membrane interactions control residue fluctuations of outer membrane porins.

Bacterial outer membrane porins (Omp) that have robust ß -barrel structures, show potential applications for nanomedicine devices in synthetic membranes and single molecule detection biosensors. Here, we explore the conformational dynamics of a set of 22 outer membrane porins, classified into five major groups: general porins, specific porins, transport Omps, poreless Omps and composed pores. Normal mode analysis, based on mechanical vibration theory and elastic network model, is performed to study the fluctuations of residues of aforementioned porins around their equilibrium positions. We find that a simple modification in this model considering weak interaction between protein and membrane, dramatically enhance the stability of results and improve the correlation coefficient between computational output and experimental results.

Interaction of luminal calcium and cytosolic ATP in the control of type 1 inositol (1,4,5)-trisphosphate receptor channels.

Ca(2+) within intracellular stores (luminal Ca(2+)) is believed to play a role in regulating Ca(2+) release into the cytosol via the inositol (1,4,5)-trisphosphate (Ins(1,4,5)P(3))-gated Ca(2+) channel (or Ins(1,4,5)P(3) receptor). To investigate this, we incorporated purified Type 1 Ins(1,4,5)P(3) receptor from rat cerebellum into planar lipid bilayers and monitored effects at altered luminal [Ca(2+)] using K(+) as the current carrier. At a high luminal [Ca(2+)] and in the presence of optimal [Ins(1,4,5)P(3)] and cytosolic [Ca(2+)], a short burst of Ins(1,4,5)P(3) receptor channel activity was followed by complete inactivation. Lowering the luminal [Ca(2+)] caused the channel to reactivate indefinitely. At luminal [Ca(2+)], reflecting a partially empty store, channel activity did not inactivate. The addition of cytosolic ATP to a channel inactivated by high luminal [Ca(2+)] caused reactivation. We provide evidence that luminal Ca(2+) is exerting its effects via a direct interaction with the luminal face of the receptor. Activation of the receptor by ATP may act as a device by which cytosolic Ca(2+) overload is prevented when the energy state of the cell is compromised.

Expression of a gene for a porin-like protein of the OmpA family from Mycobacterium tuberculosis H37Rv.

An open reading frame in the genomic database of Mycobacterium tuberculosis H37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned into Escherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for a presumed signal sequence was well expressed, but the protein had no pore-forming activity in the liposome swelling assay. However, the intact protein, OmpATb, behaved as a porin of low specific activity, with a pore diameter of 1.4 to 1.8 nm, and was also active in planar lipid bilayers, showing a single-channel conductance of 700 pS. The protein had a molecular mass of about 38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal rabbit antiserum raised to the truncated protein recognized a protein of similar molecular mass in detergent extracts of broken M. tuberculosis cells. Reverse transcription-PCR confirmed that the gene for OmpATb was expressed in M. tuberculosis cells growing in culture. Comparison of the purified protein with that in the detergent-extracted preparation using liposomes and planar lipid bilayers showed that the two materials had similar pore-forming properties. OmpATb is different from either of the mycobacterial porins described so far. This is the first report of a porin-like molecule from M. tuberculosis; the porin is likely to be important in controlling the access of hydrophilic molecules to the bacterial cell.