LIMITATIONS OF THE WHOLE CELL PATCH CLAMP TECHNIQUE IN THE CONTROL OF INTRACELLULAR CONCENTRATIONS
LIMITATIONS OF THE WHOLE CELL PATCH CLAMP TECHNIQUE IN THE CONTROL OF INTRACELLULAR CONCENTRATIONS
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DOI：
10.1016/s0006-3495(90)82418-8
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发表时间：
1990-09-01
影响因子：
3.4
通讯作者：
OLIVA, C
中科院分区：
文献类型：
--
作者：
MATHIAS, RT;COHEN, IS;OLIVA, C
最近的实验研究（Pusch和Neher，1988）和理论研究（奥利瓦等人，1988）已经发现移液管尖端是全细胞膜片钳配置中扩散的显著屏障。在本文中，我们扩展了理论分析的吸管和细胞之间的通量，包括跨膜通量。一般结论是：（a）在移液管内，离子通量主要由扩散而不是电压梯度驱动。(b)在稳定状态下，本体移液管和细胞内溶液之间存在浓度差，其由Δ c = jRp/D ρ描述，其中对于通过电阻Rp = 1 M Ω的移液管的通量j = 1fmol/s，Δ c = 1 mM，填充有电阻率ρ的溶液。= 100 Ω- cm，给定溶质扩散系数，D = 10-5 cm 2/s。(c)无论转运方向如何，达到稳态的时间总是被膜转运加速。我们应用我们的分析测量运输的Na/K泵和Na/Ca交换器在细胞从哺乳动物心脏的心室。我们发现，如果不校正细胞内和吸管Na+之间的浓度差，细胞内Na+与Na/K泵的结合曲线将显着不那么陡峭，更线性。细胞外Na+与Na/Ca交换器的结合曲线也会发生类似的变化，这是由于当交换器受到刺激时细胞内Ca++的耗尽。最后，在附录中，我们分析了移动的和固定的细胞内缓冲液对移液器和细胞之间Ca++移动的影响。固定缓冲液大大减慢了移液管和细胞内Ca++平衡的时间。移动的缓冲液的作用类似于穿梭系统，因为它们将Ca++从移液管运送到细胞，然后在它们空的时候扩散回来。Na/Ca交换剂的剧烈转运耗尽了移动的缓冲钙，从而刺激移液器的扩散以匹配Ca++转运速率。此外，我们发现，结合的Ca++的交换器可以受到影响的移动的缓冲。
Recent experimental studies (Pusch and Neher, 1988) and theoretical studies (Oliva et al., 1988) have found that the pipette tip is a significant barrier to diffusion in the whole cell patch clamp configuration. In this paper, we extend the theoretical analysis of fluxes between the pipette and cell to include transmembrane fluxes. The general conclusions are: (a) within the pipette, ion fluxes are driven primarily by diffusion rather than voltage gradients. (b) At steady state there is a concentration difference between the bulk pipette and intracellular solution that is described by .DELTA.c = jRp/D.rho., where .DELTA.c = 1 mM for a flux, j = 1 fmol/s, through a pipette of resistance, Rp = 1 M.OMEGA., filled with a solution of resistivity, .rho. = 100 .OMEGA.-cm, given a solute diffusion coefficient, D = 10-5 cm2/s. (c) The time to steady state is always accelerated by membrane transport, regardless of the direction of transport. We apply our analysis to the measurement of transport by the Na/K pump and Na/Ca exchanger in cells from the ventricles of mammalian heart. We find that the binding curve for intracellular Na+ to the Na/K pump will appear significantly less steep and more linear if one does not correct for the concentration difference between intracellular and pipette Na+. Similar shifts in the binding curve for extracellular Na+ to the Na/Ca exchanger can occur due to depletion of intracellular Ca++ when the exchanger is stimulated. Lastly, in Appendix we analyzed the effects of mobile and fixed intracellular buffers on the movement of Ca++ between the pipette and cell. Fixed buffers greatly slow the time for equilibration of pipette and intracellular Ca++. Mobile buffers act like a shuttle system, as they carry Ca++ from pipette to cell then diffuse back when they are empty. Vigorous transport by the Na/Ca exchanger depletes mobile buffered calcium, thus stimulating diffusion from the pipette to match the rate of Ca++ transport. Moreover, we find that binding of Ca++ to the exchanger can be affected by the mobile buffer.