Fein A and Terasaki M (2005), Rapid increase in plasma membrane chloride perm...

ECB-ART-39507

J Gen Physiol 2005 Aug 01;1262:151-9. doi: 10.1085/jgp.200509294.

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Rapid increase in plasma membrane chloride permeability during wound resealing in starfish oocytes.

Fein A , Terasaki M .

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Plasma membrane wound repair is an important but poorly understood process. We used femtosecond pulses from a Ti-Sapphire laser to make multiphoton excitation-induced disruptions of the plasma membrane while monitoring the membrane potential and resistance. We observed two types of wounds that depolarized the plasma membrane. At threshold light levels, the membrane potential and resistance returned to prewound values within seconds; these wounds were not easily observed by light microscopy and resealed in the absence of extracellular Ca(2+). Higher light intensities create wounds that are easily visible by light microscopy and require extracellular Ca(2+) to reseal. Within a few seconds the membrane resistance is approximately 100-fold lower, while the membrane potential has depolarized from -80 to -30 mV and is now sensitive to the Cl(-) concentration but not to that of Na(+), K(+), or H(+). We suggest that the chloride sensitivity of the membrane potential, after wound resealing, is due to the fusion of chloride-permeable intracellular membranes with the plasma membrane.

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Genes referenced: LOC100887844 LOC115919910 LOC115925415 LOC752081 LOC756768

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	Figure 1. Two distinct types of changes in membrane potential and membrane resistance caused by wounding the plasma membrane of starfish oocytes. (A) Wounding with a high light intensity causes a sustained change in the membrane potential. In the photomicrograph, there is a large hemispherical indentation where the line scan occurred (black arrowhead). The wound appears similar to wounds produced by local application of Triton X-100 detergent to the surface of sea urchin eggs, where the hemispherical boundary was shown to be continuous with the plasma membrane (McNeil et al., 2000). (B) Wounding with threshold light intensities causes a rapid depolarization of the plasma membrane, which rapidly returns to its value before wounding. In the photomicrograph there is no discernible effect of the wounding on the cell membrane in the region where it was wounded (white arrowhead). The âˆ¼50 Î¼m spherical structure inside the oocyte is the nucleus. In the records of A and B, the membrane resistance was monitored by measuring the change in membrane potential resulting from a repeated 1-nA current pulse delivered to the cell via the intracellular pipette. See text and MATERIALS AND METHODS for further experimental details. Bar, 50 Î¼m.
	Figure 2. The effect of extracellular calcium on the electrical response to wounding. (A) After wounding, the oocyte in normal Ca2+ ASW, the membrane potential is approximately âˆ’30 mV, and the membrane resistance is below the resolution of the bridge monitor. (B) After wounding the oocyte in 0 Ca2+ 1 mM EGTA ASW, the membrane potential slowly goes to approximately zero and the membrane resistance is below the resolution of the bridge monitor. At the end of each experiment in A and B, the intracellular pipette was withdrawn from the cell into the bath to confirm the value of the membrane potential. See text for further experimental details.
	Figure 3. Chloride ion permeability contributes to the membrane potential following wounding. (A) The cell was bathed in Na isethionate ASW and subsequently wounded. At the end of the experiment, the intracellular pipette was withdrawn from the cell into the bath to confirm the value of the membrane potential. (B) Effect of ion substitution on the membrane potential (mean Â± SD) following wounding. Control, ASW (n = 10), 100 mM K+ ASW (n = 6), choline ASW (n = 7), pH 7.0 ASW (n = 6), Na isethionate ASW (n = 9). (C) Clâˆ’ dependence of the membrane potential after wounding. The data were fit with a straight line solely for the purpose of providing a quantitative estimate of the chloride dependence of the membrane potential after wounding. (D) Superimposition of the recordings of Fig. 1 A and Fig. 3 A aligned at the time of wounding. For oocytes bathed in 100 mM K+, the resting membrane potential before wounding was âˆ’21.1 Â± 1.4 mV (n = 6 cells) because the plasma membrane of mature and immature starfish oocytes exhibit an inwardly rectifying K+ current that predominately sets the resting membrane potential (for example see Shen and Steinhardt, 1976).
	Figure 4. Simultaneous electrical recording and fluorescence imaging during wound resealing. Oocytes were immersed in calcein-containing sea water and imaged before and after plasma membrane disruption while the membrane potential and membrane resistance of the oocyte were monitored simultaneously. Each image was obtained at the corresponding times indicated on the electrical recordings below. The dark spot at the wound site, which shrinks over time, and is just below where the membrane was, is a gas bubble that sometimes forms after wounding. Bar, 50 Î¼m.
	Figure 5. Effect, on wound healing, of removing the oocyte extracellular matrix. The vitelline envelope was removed by protease treatment (see MATERIALS AND METHODS). The oocyte was immersed in sea water containing 0.5 mg/ml calcein and wounded using the same wounding protocol as in Fig. 4. Fluorescence (bottom) and transmitted light (top) images were obtained simultaneously. The first time point shown was taken just before the wound. The second time point shown is the first image taken after the wound, and the other time points are the successive images taken at 0.985-s intervals. In contrast to Fig. 1 A, where the vitelline envelope was present, the cytoplasm bulges outward. Very little calcein appears to enter the cell. Extracellular debris can be seen, particularly as negative fluorescence images; this debris may arise from the yolk granule contents released into the sea water after fusion with the plasma membrane. Bar, 10 Î¼m.
	Figure 6. Extracellular calcium is not required for recovery from wounds caused by threshold light intensities. (A) A threshold light causes a rapid depolarization of the plasma membrane, which rapidly returns to its value before wounding. Similarly the membrane resistance after wounding is approximately the same as it was before the wound. (B) Membrane current measured under voltage clamp during wounding resulting from threshold intensity multiphoton excitation. The cell was impaled with two microelectrodes and placed under voltage clamp at a holding potential of âˆ’30 mV. The membrane potential was repeatedly ramped from âˆ’35 to âˆ’25 mV in order to monitor the slope resistance of the plasma membrane around âˆ’30 mV, see upper membrane potential trace. Wounding the cell caused a transient inward current having peak amplitude of âˆ¼6 nA (see lower membrane current trace). The cells in A and B were bathed in 0 Ca2+ 1 mM EGTA ASW throughout the experiment. See text for further experimental details. We follow the convention of showing inward currents as a downward deflection.
	Figure 7. Simultaneous electrical recording and fluorescence imaging during wound resealing in calcium-free sea water. Oocytes were immersed in calcein-containing CFSW and imaged before and after plasma membrane disruption while the membrane potential and membrane resistance of the oocyte were monitored simultaneously. Each image was obtained at the corresponding times indicated on the electrical recordings below. Bar, 10 Î¼m.

References [+] :

Bansal, Defective membrane repair in dysferlin-deficient muscular dystrophy. 2003, Pubmed