BENER Abstract No. 15058. INHIBITION OF CALCIUM CHANNEL ACTIVATION IN GH3 CELLS BY STATIC MAGNETIC FIELDS.
(Eng.) Rosen, A. D. [Dept. of Neurology, Sch. of Medicine, State Univ. of New York, Stony Brook, NY 11794-8121; Electronic mail: arosen@neuro.som.sunysb.edu] Biochim Biophys Acta 1282(1):149-155; 1996 (25 Refs).
A whole-cell patch-clamp method was used with cultured GH3 cells to examine calcium channel function in the presence of static magnetic fields (SMF). Proliferating GH3 cells were grown in 100-mm polystyrene culture dishes at 37 C in a 5% CO2 environment. The growth medium contained DMEM supplemented with 10% fetal calf serum and penicillin/streptomycin. As the cells approached confluency, they were split and replated at 10% of their initial density. The electromagnet used in the study consisted of a 2,700-turn coil wound on a 2.4-cm2 soft-iron C-core with an air gap of 44 mm. The coil was powered by a computer-controlled current source that could generate static fields with flux densities to 125 mT (1.25 kG). The magnet was mounted on an aluminum stage that fit a Zeiss Axiovert 100 microscope. Recordings on the cells were made within the 650-um diameter fixed microscope field, centered midway between the magnet poles. The growth medium was slowly exchanged with a recording solution consisting of 120 mM NaCl, 10 mM CaCl2, 2 mM MgCl2, 1 uM TTX, and 10 mM HEPES titrated with NaOH to a pH of 7.2. Electrodes were filled with a solution consisting of 120 mM CsCl, 10 mM K-EGTA, and 10 mM HEPES titrated with KOH to a pH of 7.2. Use of TTX abolished inward sodium current, and outward potassium current was eliminated by replacing intracellular K+ with Cs+. The EGTA chelated Ca++ and consequently inhibited Ca++-activated potassium channels. Membrane current was recorded with an Axopatch 1-D patch clamp amplifier, the command voltages of which were generated with HEKA Pulse software running on a Macintosh 840AV computer. Current responses were digitized at 40 kHz on an Instrutech ITC-16 A/D converter and processed for display by the Pulse software. Once the system was stable, a 120-mT (1.2 kG) SMF was applied for 150 sec. The voltage jump sequence was repeated beginning 100 sec after the field was turned on and at 30, 100, and 170 sec after it was turned off. The currents were measured in 37 cells, 15 um in diameter, using the whole-cell configuration of the patch clamp technique. During exposure at 120 mT SMF, a 10-mV shift was observed in the current-voltage relationship and a 143-pA reduction in the peak current amplitude. The most significant change occurred in the slowing of the activation rate, observed on examination of the calcium current tracings. An increase in the calcium channel activation time constant without significant change in the inactivation time constant was clearly observed. This increase was found to be temperature dependent and was present at all levels of membrane depolarization, but was greatest between -35 and -15 mV, where it more than doubled. This was the most consistent change during SMF exposure and was fully reversible 2-3 min after the field was turned off. The highly ordered phospholipid bilayer structure of biological membranes present substantial diamagnetic properties. At higher temperatures, an abrupt rotameric disordering of the lipid acyl chains occurs with a transition to a liquid-crystal phase and a less rigid membrane, which is more readily deformable by a magnetic filed. Such a deformity could alter the function of imbedded calcium channels. However, the portion of the channel outside of the membrane per say will be unaffected. Additional mechanisms such as changes in the function of potassium or sodium channels may also be operative.
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