title: Limitations of the whole cell patch clamp technique in the control of intracellular concentrations.
abstract: 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/Dp, 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, p = 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 analyze 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.
authors.affiliation: Department of Physiology and Biophysics, State University of New York, Stony Brook 11794.
authors.forename: R T
authors.initials: RT
authors.lastname: Mathias
authors.forename: I S
authors.initials: IS
authors.lastname: Cohen
authors.forename: C
authors.initials: C
authors.lastname: Oliva
mesh_terms: D000818:Animals; D001693:Biological Transport, Active; D002021:Buffers; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D002463:Cell Membrane Permeability; D005069:Evaluation Studies as Topic; D008954:Models, Biological; D009206:Myocardium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
chemical_list: D002021:Buffers; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium; D002118:Calcium