The influence of electrochemical gradients of Na+ and K+ upon the membrane binding and pore forming activity of the terminal complement proteins

Sims, P.; Wiedmer, Therese

doi:10.1007/bf01869204

Cited by 10 publications

(7 citation statements)

References 32 publications

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“…Similarly, applied voltages of 10-60 mV during addition of the C5b-9 proteins did not substantially affect the resulting membrane conductance, suggesting that C5b-9 assembly occurs independently of Vm. A similar conclusion was reached previously on the basis of direct measurement of C5b-9 binding to valinomycin-treated erythrocyte membranes (Sims & Wiedmer, 1984a). Unfortunately, it was not possible to perform these experi- …”

Section: Current-voltage Relationshipsupporting

confidence: 61%

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

et al. 1986

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Single-channel analysis of electrical fluctuations induced in planar bilayer membranes by the purified human complement proteins C5b6, C7, C8, and C9 have been analyzed. Reconstitution experiments with lipid bilayer membranes showed that the C5b-9 proteins formed pores only if all proteins were present at one side of the membrane. The complement pores had an average single-channel conductance of 3.1 nS at 0.15 M KCl. The histogram of the complement pores suggested a substantial variation of the size of the single channel. The linear relationship between single-channel conductance at fixed ionic strength and the aqueous mobility of the ions in the bulk aqueous phase indicated that the ions move inside the complement pore in a manner similar to the way they move in the aqueous phase. The minimum diameter of the pores as judged from the conductance data is approximately 3 nm. The complement channels showed no apparent voltage control or regulation up to transmembrane potentials of 100 mV. At neutral pH the pore is three to four times more permeable for alkali ions than for chloride, which may be explained by the existence of fixed negatively charged groups in or near the pore. The significance of these observations to current molecular models of the membrane lesion formed by these cytolytic serum proteins is considered.

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Section: Current-voltage Relationshipsupporting

confidence: 61%

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

et al. 1986

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“…2 and 3) even after prolonged (2-hr) incubation (data not shown; see also Dalmasso et al, 1975;Sims & Wiedmer, 1984). These results suggest that although depolarization of Em after C5b-9 binding is a necessary antecedent to cell lysis, this electrochemical change is not invariably sufficient to achieve cytolytic rupture of the plasma membrane.…”

Section: Discussionmentioning

confidence: 61%

“…When exposed to identical concentrations of C8 and C9, the onset of the fluorescence response of EACl-7, which presumably signals depolarization of the membrane potential to the 0 mV level, occurred substantially faster when the cells were suspended in KCI-MOPS versus NaCI-MOPS, coinciding with the increased complement-mediated hemolysis that is also observed for these high potassium-containing erythrocytes when suspended in KC1 versus NaC1 (Fig. 2, and Dalmasso et al, 1975;Sims & Wiedmer, 1984). A close inspection of the traces of Fig.…”

Section: Membrane Potential Changes Initiated By the Binding Of C8 Anmentioning

confidence: 80%

“…The possibility that this putative transmembrane channel might selectively conduct potassium (versus sodium) was suggested by: (i) the enhanced lysis observed when C5b-9-treated erythrocytes are suspended in isotonic KC1 versus NaC1, which we have recently shown reflects an effect on the target cell which is exerted after the binding of the C5b-9 proteins (Dalmasso et al, 1975;Sims & Wiedmer, 1984); (ii) the differential effect of anisoosmotic solutions of KC1 versus NaC1 on the kinetics of complement-mediated hemolysis (Lauf, 1975;1978); (iii) data pertaining to the molecular exclusion size of the C5b-9 pore, which suggest a minimum pore radius of <4 A (Sims & Lauf, 1978;1980); (iv) recent analysis of the single-channel conductance associated with binding of the C9b fragment of complement protein C9 to lipid bilayer membranes, which indicates a K/Na selectivity ratio of 3.5 : 1 (Esser et al,, 1984).…”

Section: Discussionmentioning

confidence: 97%

“…Resealed ghost membranes of human erythrocytes (ghosts) were prepared by reversible hypotonic lysis at 0~ using a modification of published procedures (Sims & Lauf, 1978: Sims & Wiedmer, 1984. Lysis of 1 vol red cell suspension (2 x 109/ml) was performed at pH 6.0 to 6.1 in 10 vol 4 mM MgSO4, 2 mM ATP.…”

Section: Resealed Erythrocyte Ghostsmentioning

confidence: 99%

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Cyanine dye fluorescence used to measure membrane potential changes due to the assembly of complement proteins C5b-9

Wiedmer

Sims

1985

J. Membrain Biol.

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The fluorescent potentiometric indicator diS-C3-(5) has been used to investigate changes in membrane potential due to assembly of the C5b-9 membrane attack complex of the complement system. EAC1-7 human red blood cells and resealed erythrocyte ghosts--bearing membrane-assembled C5b67 complexes--were generated by immune activation in C8-deficient human serum. Studies performed with these cellular intermediates revealed that the membrane potential of EAC1-7 red cells and ghosts is unchanged from control red cells (-7 mV) and ghosts (O mV), respectively. Addition of complement proteins C8 and C9 to EAC1-7 red cells results in a dose-dependent depolarization of membrane potential which precedes hemolysis. This prelytic depolarization of membrane potential--and the consequent onset of hemolysis--is accelerated by raising external [K+], suggesting that the diffusional equilibration of transmembrane cation gradients is rate limiting to the cytolytic event. In the case of EAC1-7 resealed ghosts suspended at either high external [K+] or [Na+], no change in membrane potential (from O mV) could be detected after C8/C9 additions. When the membrane potential of the EAC1-7 ghost was displaced from O mV by selectively increasing the K+ conductance with valinomycin, a dose-dependent depolarization of the membrane was observed upon addition of C8 and C9. In these experiments, lytic breakdown of the ghost membranes was less than 5%. Conclusions derived from this study include: (i) measured prelytic depolarization of the red cell Donnan potential directly confirms the colloid-osmotic theory of immune cytolysis. (ii) The diffusional transmembrane equilibration of Na+ and K+ through the C5b-9 pore results in a dose-dependent depolarization of the membrane potential (Em) which appears to be rate-limiting to cytolytic rupture of the target erythrocyte. (iii) Enhanced immune hemolysis observed in high K+ media cannot be attributed to cation-selective conductance across the C5b-9 pore, and is probably related to the near-equilibrium condition of potassium-containing red cells when suspended at high external K+. These experiments demonstrate that carbocyanine dye fluorescent indicators can be used to monitor electrochemical changes arising from immune damage to the plasma membrane under both cytolytic and noncytolytic conditions. Potential application of this method to the detection of sublytic pathophysiological changes in the plasma membrane of complement-damaged cells are discussed.

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Complement proteins C5b-9 induce transbilayer migration of membrane phospholipids

Meer¹,

Fugate²,

Sims³

1989

Biophysical Journal

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Transbilayer migration of membrane phospholipid arising from membrane insertion of the terminal human complement proteins has been investigated. Asymmetric vesicles containing pyrene-labeled phosphatidylcholine (pyrenePC) concentrated in the inner monolayer were prepared by outer monolayer exchange between pyrenePC-containing large unilamellar vesicles and excess (unlabeled) small unilamellar vesicles, using bovine liver phosphatidylcholine-specific exchange protein. After depletion of pyrenePC from the outer monolayer, the asymmetric large unilamellar vesicles were isolated by gel filtration and exposed to the purified C5b-9 proteins at 37 degrees C. Transbilayer exchange of phospholipid between inner and outer monolayers during C5b-9 assembly was monitored by changes in pyrene excimer and monomer fluorescence. Membrane deposition of the C5b67 complex (by incubation with C5b6 + C7) caused no change in pyrenePC fluorescence. Addition of C8 to the C5b67 vesicles resulted in a dose-dependent decrease in the excimer/monomer ratio. This change was observed both in the presence and absence of complement C9. No change in fluorescence was observed for control vesicles exposed to C8 (in the absence of membrane C5b67), or upon C5b-9 addition to vesicles containing pyrenePC symmetrically distributed between inner and outer monolayers. These data suggest that a transbilayer exchange of phospholipid between inner and outer monolayers is initiated upon C8 binding to C5b67. The fluorescence data were analyzed according to a "random walk" model for excimer formation developed for the case where pyrenePC is asymmetrically distributed between lipid bilayers. Based on this analysis, we estimate that a net transbilayer migration of approximately 1% of total membrane phospholipid is initiated upon C8 binding to C5b67. The potential significance of this transbilayer exchange of membrane phospholipid to the biological activity of the terminal complement proteins is considered.

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The influence of electrochemical gradients of Na+ and K+ upon the membrane binding and pore forming activity of the terminal complement proteins

Cited by 10 publications

References 32 publications

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

Cyanine dye fluorescence used to measure membrane potential changes due to the assembly of complement proteins C5b-9

Complement proteins C5b-9 induce transbilayer migration of membrane phospholipids

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