Multiple ion binding sites in I_h channels of rod photoreceptors from tiger salamanders

The mechanism of ion permeation in K⁺/Na⁺-permeable I_h channels of tiger salamander rod photoreceptors was investigated using the whole-cell voltage-clamp technique. I_h channels showed features indicative of pores with multiple ion binding sites: in mixtures of K⁺ and thallium Tl⁺, the amplitude of the time-dependent current showed an anomalous mole fraction dependence, and K⁺ permeation was blocked by other permeant ions (with K_0.5 values: Tl⁺, 44 μM; Rb⁺, 220 μM and NH₄⁺, 1100 /gmM) as well as by essentially impermeant ions (Cs⁺, 22 μM Ba²⁺, 9200 μM) which apparently block I_h by binding in the pore. In contrast, Na⁺ had little blocking action on K⁺ permeation. The block by all of these ions was sensitive to external K⁺ with the block by Cs⁺ being the least sensitive. Na⁺ was more effective than K⁺ in reducing the block by Tl⁺, Rb⁺ and NH₄⁺, but was less effective for the block by Cs⁺ and Ba²⁺. The blocking action of Cs⁺ and Ba²⁺ was non-competitive, suggesting that they block I_h channels at independent sites. Based on the efficacy of block by the different ions, the degree to which K⁺ and Na⁺ antagonize this block and the noncompetitive blocking action of Cs⁺ and Ba²⁺, the permeation pathway of I_h channels appears to contain at least three ion binding sites with at least two sites having a higher affinity for K⁺ over Na⁺ and another site with a higher affinity for Na⁺ over K⁺.