Surface potentials near the mouth of the large-conductance K channel from Chara australis: A new method of testing for diffusion-limited ion flow.

The kinetics of single K channels were derived for patch-clamp recordings of membrane patches excised from cytoplasmic drops from the plant, Chara australis R. Br. Specifically, the 'tilt effect' model of MacKinnon, Latorre and Miller (1989. Biochemistry 28:8092-8099) has been used to measure the electrostatic potential (surface PD) and fixed charge at the entrances of the channel. The surface PD is derived from the difference between the trans-pore potential difference (PD) and that between the two bulk phases. The trans-pore PD is probed using three voltage-dependent properties of the channel. These are (1) the association and dissociation rates of Ca binding to the channel, from both the cytoplasmic and vacuolar solutions. These were determined from the mean blocked and unblocked durations of the channel in the presence of either 20 mmol liter vacuolar or 1 mmol liter cytoplasmic Ca; (2) the closing rate of the channel's intrinsic gating process. This was determined from the mean channel open time in the absence of vacuolar Ca at membrane PDs more negative than −100 mV; and (3) the effect of Mg on channel conductance when added to solutions initially containing 3 mmol liter KCl. The voltage dependence of properties 1 and 2 shifts along the voltage axis according to the ionic strength of the bathing media, consistent with the presence of negative charge in the channel vestibules. Furthermore, the magnitude of this shift depends on the current in a manner consistent with diffusion-limited ion flow in the channel (i.e., the rate of ion diffusion in the external electrolyte limits the channel conductance). Mg on either side of the membrane alters channel conductance in a voltage-dependent way. A novel feature of the Mg effect is that it reverses, from a block to an enhancement, when the membrane PD is more negative than −70 mV. This reversal only appears in solutions of low ionic strength. The attenuating effect is due to voltage-dependent binding of Mg within the pore, which presumably plugs the channel. The enhancing effect is due to screening by Mg of surface potentials arising from diffusion-limited flow of K. All experimental approaches give a consistent picture of K permeation in which the surface charge and convergence permeability of the cytoplasmic vestibule are the major factors in determining channel conductance. The cytoplasmic vestibule has a charge density of −0.035 C/m which is similar to that found for maxi K channels in rat muscle. The properties of the vacuolar vestibule, which is effectively neutral, differ from the negatively charged external vestibules in rat maxi K channels indicating a differing protein structure in this part of the channel. Finally, we note that our method of testing for diffusion-limited ion flow, by measuring the dependence of the surface PD on the current passing through the channel, is more reliable than common tests, which make use of nonelectrolytes such as sucrose. It appears that these molecules alter channel conductance by interfering with the intrinsic permeation mechanism of the channel rather than by altering bulk viscosity. [ABSTRACT FROM AUTHOR]