Movement through the Pore in IRK1 Potassium Channels

We measured unidirectional K 1 inand 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 1 concentration of 25 mM, the data were described by a Ussing flux-ratio exponent, n 9 , of z 2.2 and was constant over a voltage range from 2 50 to 2 25 mV. This result indicates that the pore of IRK1 channels may be simultaneously occupied by at least three ions. The IRK1 n 9 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 1 concentration. The conductance could be fit by a saturating hyperbola with a half-saturation K 1 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 1 selectivity and multi-ion occupancy, they differ in other properties, including the sensitivity of pore conductance to K 1 concentration, and may differ in the number of K 1 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. key words: ion permeation • unidirectional flux-ratio • single channel conductance i n t r o d u c t i o n The permeation properties of K channels are quite complex and are not consistent with the independent movement of ions through simple aqueous pores. Rather, these complex properties suggest that the channel pore may simultaneously be occupied by several ions. Some of the properties of ion channels that are consistent with multi-ion pore occupancy include: ( a ) an apparent concentration-dependent ion selectivity, ( b ) a voltage sensitivity of pore block by ions that depends on the concentration of the permeant ion or the blocking ion (or both), ( c ) pore current (or conductance or permeability ratio) that is a nonmonotonic function of the mole fraction of two types of ions (the so-called “anomalous mole-fraction effect”), and ( d ) deviations from the Ussing (1949) flux ratio test for independent ion movement. This latter test, as applied by Hodgkin and Keynes (1955), relies on a modified form of the Ussing flux ratio test for independent ion movement: , (1) where m e and m i are unidirectional K 1 efflux and influx, respectively, [K] i and [K] o are the intracellular and extracellular K 1 concentrations, V m is the membrane voltage, and R, T , and F have their usual thermodynamic meanings. The flux-ratio exponent, n 9 , is 1 for a pore in which ion movements are independent; that is, a pore that never contains more than a single ion. The flux-ratio test has been applied to several types of K channels in native cells, including delayed rectifier channels in cephalopod axons (Hodgkin and Keynes, 1955; Begenisich and De Weer, 1980), the Ca 2 1 -activated K channel in erythrocytes (Vestergaard-Bogind et al., 1985), and the inward rectifier K channel in skeletal muscle (Horowicz et al., 1968; Spalding et al., 1981). In all these cases, the flux-ratio exponent was found to be significantly . 1.0, demonstrating the multi-ion nature of these types of channels. We recently determined the flux-ratio exponent for cloned Shaker K channels expressed in Xenopus oocytes (Stampe and Begenisich, 1996). We found a value of z 3.5 at 2 30 mV, which is essentially the same as that from native squid axon K channels (Begenisich and De Weer, 1980). The largest n 9 value for native inward-recme mi ----K [ ] i K [ ] o ------------exp VmF RT ---------    n ′ = Address correspondence to Ted Begenisich, Department of Pharmacology & Physiology, Box 711, University of Rochester Medical Center, Rochester, NY 14642. Fax: 716-244-9283; E-mail: tbb@crocus. medicine.rochester.edu on Jne 0, 2017 D ow nladed fom Published October 1, 1998