SUMMARY1. The hyperpolarization between bursts in the R 15 cell of Aplysia is accompanied by an increase in membrane slope conductance.2. The post-burst hyperpolarization can be observed with ouabain, lithium, or potassium-free solution if artificial inward current is applied. The hyperpolarization can be observed with dinitrophenol or cooling to 10°C, with no injected current. Thus, the hyperpolarization apparently is not due to the cyclic activity of an electrogenic pump.3. A reversal potential for the post-burst hyperpolarization can be demonstrated by passage of inward current during the inter-burst period. The reversal of direction of the potential depends on recent occurrence of a burst.4. The reversal potential varies with external potassium concentration, but not with chloride or sodium.5. The post-burst hyperpolarization is not blocked by external tetraethylammonium at a concentration which greatly prolongs the action potentials.6. During the onset of spike blockage by, and recovery from, calciumfree + tetrodotoxin saline, the bursts of action potentials appear to be driven by endogenous waves of membrane potential.7. The hyperpolarizing phase of the waves in calcium-free + tetrodotoxin medium is accompanied by an increased slope conductance.8. A reversal potential can be demonstrated for the hyperpolarization following a wave in calcium-free + tetrodotoxin medium by applying inward current during the interwave period.9. The waves in calcium-free + tetrodotoxin medium are blocked by ouabain but can be reinstated by artificial hyperpolarization.10. The post-burst hyperpolarization and the post-wave hyperpolarization appear to result from a periodic increase in membrane conductance, primarily to potassium ions. DOUGLAS JUNGE AND CATHY L. STEPHENS
An extracellular patch electrode was used to record ionic currents from individual complement-induced channels in the membranes of antibody-coated skeletal muscle. The amplitude of the single-channel currents leads to an estimate of 90 pS for the unit conductance. The kinetics of channel opening and closing show marked variability and complexity. Channels flicker open and closed repeatedly, indicating that once these lesions form, they undergo rapid structural transitions between discrete conducting and nonconducting states.Activation ofcomplement initiates a cascade ofevents whereby several protein components combine to damage a cell membrane. Solute molecules pass through the damaged membrane to create an osmotic imbalance which leads to cytolysis (1). Complement-induced damage has been demonstrated in synthetic lipid bilayers, where it is accompanied by discrete conductance increases (2-5). We report here the measurement of electrical current through individual molecular-size lesions produced by complement in the membranes of intact muscle cells grown in tissue culture. These experiments use the recently developed patch-electrode technique for isolating the electrical current passing through a very small area ofcell membrane (6). This patch recording method has been used to observe single ionic channel currents in synaptic (7-9) and axonal (10, 11) membranes. Signals observed in this way allow us to estimate the conductance of the channel and to characterize the kinetic behavior of channel opening and closing. METHODS Antibody-coated muscle was prepared as described by Stephens and Henkart (12). Cultured rat muscle is first treated with 1 mM trinitrobenzylsulfonic acid in Dulbecco's phosphate-buffered saline (referred to as saline) for 30 min at room temperature. This results in the nonselective attachment ofthe hapten 2,4,6-trinitrophenol (TNP) to membrane proteins. After washing off the trinitrobenzylsulfonic acid, the cells are bathed in purified antibody against TNP for 45 min.A patch electrode, filled with rabbit complement diluted up to 1:3 with saline, is pressed against the cell surface as illustrated in Fig. 1A. This allows the antibody-hapten conjugates under the tip ofthe electrode to activate complement in the electrode. The activated complement then attacks the membrane under the electrode tip to produce current fluctuations as shown in Fig. 1B.The patch electrode is fabricated to seal tightly with the cell membrane, confining the complement attack to the 1-to 2-,um2 In one experiment, complement was inactivated by prior activation. A crude preparation of TNP-labeled bovine serum albumin was prepared by mixing 1 mM trinitrobenzylsulfonic acid with 0. 15mM albumin. After standing at room temperature for 4 hr, the mixture was diluted 1:1000 (vol/vol) with saline.One aliquot was mixed with anti-TNP antibody during the dilution so that the final antibody concentration was 0.67 LM. TNP-labeled albumin with and without antibody was mixed 1:1 (vol/vol) with complement and allowed to stand at r...
Cultered neuroblastoma-glioma hybrid cells and primary mouse and rat muscle cells were studied by using intracellular microelectrodes to monitor membrane electrical potential and resistance changes during complement-mediated lysis. The cell membrane was TNP modified under mild conditions and subsequently coated with rabbit IgG anti-TNP, with no electrical changes observed. However, upon addition of guinea pig C the membrane potential dropped from ∼-50 mv to <-12 mv within a few minutes, with parallel decreases in electrical resistance. Ten to 60 min after these electrical changes the cells became stainable with trypan blue. No electrical changes or trypan blue staining was observed in the absence of antibody or with heat-inactivated C. With more dilute C so that only a fraction of cells became trypan blue positive, all cells nevertheless showed the membrane electrical changes; surviving cells recovered their original membrane properties within 1 hr. Thus the C-mediated damage to the membrane measured electrically is not in itself sufficient to ensure the subsequent death of the cell. The early electrical changes observed appear to be comparable to increases in 86Rb efflux measured by others.
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