Unidirectional K+ fluxes through recombinant Shaker potassium channels expressed in single Xenopus oocytes.

Abstract

A B S T R A C T We describe a method to evaluate the ratio of ionic fluxes through recombinant channels expressed in a single Xenopus oocyte. A potassium channel encoded by the Drosophila Shakergene tested by this method exhibited flux ratios far from those expected for independent ion movement. At a fixed extracellular concentration of 25 mM K +, this channel showed single-file diffusion with an Ussing flux-ratio exponent, n', of 3.4 at a membrane potential of -30 mV. There was an apparent, small voltage dependence of this parameter with n' values of 2.4 at -15 and -5 inV. These results indicate that the pore in these channels can simultaneously accommodate at least four K + ions. If each of these K + ions is in contact with two water molecules, the minimum length of the pore is 24 A.

Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport

“…6B). It is important to note that, in contrast with GSH, oocytes have an active endogenous taurocholate export mechanism (31) and that the baseline rate of taurocholate export is relatively high (31) (Fig. 6B).…”

“…Single oocytes were placed in a small (0.2 ml) Plexiglas chamber and superfused with modified Barth's solution or a high-K ϩ modified Barth's solution in which the NaCl was replaced with KCl (88 mM). Membrane potential determinations were made as previously described using a custom-made voltage-clamp amplifier (31). Both reference electrodes and intracellular recording electrodes were filled with 3 M KCl, and oocytes were impaled at the vegetal pole.…”

Kcsa

“…In KcsA, the high concentration creep is of much larger amplitude in K + than in Rb + , as though the pore occupancy is on average higher for K + , possibly reflecting the channel's evolutionary fine-tuning for rapid passage of K + (rather than Rb + ) at physiological ion concentrations. In light of the long-held view based on flux-ratio exponents (Hodgkin and Keynes 1955; Begenisich and DeWeer 1980; Spalding et al 1981; Stampe and Begenisich 1996; Stampe et al 1998) that three K + ions occupy the pore under physiological conditions, this picture is hardly novel, but merely represents a natural reading of K + conduction in a structurally explicit context. Speculations of this kind must await direct tests by quantitative crystallographic measurements of ion occupancy in KcsA, which are now nearing feasibility (Jiang and MacKinnon 2000).…”

Nonequilibrium Thermodynamics of Ion Flux through Membrane Channels

“…When an ion channel and its adjacent solutions are regarded as a small nonequilibrium system, the ratio of the probability of the forward transport event (down the direction of electrochemical potential gradient) to the backward transport event (against the direction of electrochemical potential gradient) can be derived based on the fluctuation theorem [12,21]. This probability ratio is equal to the Ussing flux ratio, which is the ratio of unidirectional outward flux to unidirectional inward flux, as determined by radioisotope experimental studies in squid axon systems in the 1950s [22,23], and later in the cloned expression systems of ion channels [24,25].…”

“…The flux-ratio exponent, n, was measured to be 2.5 to 3.5 in different K + channels [22,24,25], which suggested that there might be 2 to 3 K + ions simultaneously occupying the multi-ion single file K + channel pore. The first X-ray crystal structure of the K + channel pore was determined by MacKinnon's group in 1998 [26], which confirmed the prediction by Hodgkin and Keynes.…”