The cardiac muscarinic K⁺ channel, Kir3.4-Kir3.4, is a heterotetramer and its pore is asymmetric (Silverman et al. 1998). A threonine residue is present in the Kir3.4 subunit (Kir3.4-T148), but not in the Kir3.1 subunit; in Kir3.1 an alanine residue is present instead (Kir3.1-A142). In the Kir2.1 channel, which is homotetramer, the equivalent threonine residue (T141) has been shown to be important in Cs⁺ block (Thompson et al. 2000). In Kir2.1, the mutation T141A increases the K_D for Cs⁺ block at ~-100 mV 50-fold. To test whether Kir3.4-T148 is important for block and permeation of Kir3.1-Kir3.4, we have investigated Cs⁺ block and Rb⁺ and polyamine permeation of the mutant Kir3.1-Kir3.4-T148A channel.

Kir3.1, Kir3.4 (rat clones) and hD2 (human dopamine D2 receptor) were expressed in Xenopus oocytes and ionic current was measured with the two-microelectrode voltage clamp technique in the presence of 10 mM dopamine and 90 mM extracellular K⁺.

Extracellular Cs⁺ blocked the wild-type channel with a K_D at -130 mV of 177.5 ± 4.2 µM (mean ± S.E.M.; n = 6), while the K_D of the mutant Kir3.1-Kir3.4-T148A channel was 444 ± 28 µM (n = 5); the block exhibited voltage dependence (δ = 0.8 and 0.6 for the wild-type and mutant channels, respectively). This mutation resulted only in 2.5-fold decrease in the channel sensitivity to Cs⁺ block. In the mutant Kir3.1-A142T-Kir3.4 channel, another two threonine residues have been added to the channel pore. Extracellular Cs⁺ blocked the mutant channel with a K_D at -130 mV of 443.6 ± 64 (n = 7); the block exhibited a pronounced voltage dependence (δ = 2.5). To test whether the wild-type and mutant channels are permeable to Rb⁺ and polyamine, the 90 mM K⁺ in the bathing solution was replaced by 90 mM RbCl or spermine chloride. The wild-type channel was slightly permeable to Rb⁺ and spermine: with Rb⁺ and spermine, current at -130 mV was, respectively, 0.30 ± 0.02 times (n = 5) and 0.36 ± 0.04 times (n = 5) that carried by K⁺. The mutant Kir3.1/Kir3.4-T148A channel was markedly more permeable to both Rb⁺ and polyamine: with Rb⁺ and spermine, current at -130 mV was, respectively, 0.58 ± 0.03 times (n = 6) and 1.27 ± 0.20 times (n = 6) that carried by K⁺.

These results support the hypothesis that the threonine residue in the pore region of the muscarinic K⁺ channel is important for channel block and permeation.