The external vestibules of many K+ channels carry a high-affinity receptor for charybdotoxin, a peptide of known structure. Point mutations of a recombinant toxin identified the residues directly involved in the interaction with a Ca(2+)-activated K+ channel. The interaction surface is formed from 8 of the 37 residues and covers about 25% of the peptide's molecular surface. The shape of the toxin permits a deduced picture of the complementary receptor site in the external vestibule of the K+ channel.

The ratio between the unidirectional fluxes through the Ca2+-activated K+-specific ion channel of the human red cell membrane has been determined as a function of the driving force (Vm-EK). Net effluxes and 42K influxes were determined during an initial period of approximately 90 sec on cells which had been depleted of ATP and loaded with Ca. The cells were suspended in buffer-free salt solutions in the presence of 20 microM of the protonophore CCCP, monitoring in this way changes in membrane potential as changes in extracellular pH. (Vm-EK) was varied at constant EK by varying the Nernst potential and the conductance of the anion and the conductance of the potassium ion. In another series of experiments EK was varied by suspending cells in salt solutions with different K+ concentrations. At high extracellular K+ concentrations both of the unidirectional fluxes were determined as 42K in- and effluxes in pairs of parallel experiments. Within a range of (Vm-EK) of -6 to 90 mV the ratio between the unidirectional fluxes deviated strongly from the values predicted by Ussing's flux ratio equation. The Ca2+-activated K+ channel of the human red cell membrane showed single-file diffusion with a flux ratio exponent n of 2.7. The magnitude of n was independent of the driving force (Vm-EK), independent of Vm and independent of the conductance gK.

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.

We measured unidirectional K+ in- and efflux through an inward rectifier K channel (IRK1) expressed in Xenopus oocytes. The ratio of these unidirectional fluxes differed significantly from expectations based on independent ion movement. In an extracellular solution with a K+ concentration of 25 mM, the data were described by a Ussing flux-ratio exponent, n′, of ∼2.2 and was constant over a voltage range from −50 to −25 mV. This result indicates that the pore of IRK1 channels may be simultaneously occupied by at least three ions. The IRK1 n′ value of 2.2 is significantly smaller than the value of 3.5 obtained for Shaker K channels under identical conditions. To determine if other permeation properties that reflect multi-ion behavior differed between these two channel types, we measured the conductance (at 0 mV) of single IRK1 channels as a function of symmetrical K+ concentration. The conductance could be fit by a saturating hyperbola with a half-saturation K+ activity of 40 mM, substantially less than the reported value of 300 mM for Shaker K channels. We investigated the ability of simple permeation models based on absolute reaction rate theory to simulate IRK1 current–voltage, conductance, and flux-ratio data. Certain classes of four-barrier, three-site permeation models are inconsistent with the data, but models with high lateral barriers and a deep central well were able to account for the flux-ratio and single channel data. We conclude that while the pore in IRK1 and Shaker channels share important similarities, including K+ selectivity and multi-ion occupancy, they differ in other properties, including the sensitivity of pore conductance to K+ concentration, and may differ in the number of K+ ions that can simultaneously occupy the pore: IRK1 channels may contain three ions, but the pore in Shaker channels can accommodate four or more ions.