[View Larger Version of this Image]

Figure S1. The saturating behavior of the R666G gating pore current is inconsistent with a model of divalent cation block. Divalent cation block as a potential explanation for the saturation of K⁺ currents was explored in A, in which colored lines represent best fits of the divalent block model to the data recorded in symmetrical K⁺ (red) or an inwardly biased K⁺ gradient (blue). Data points are replicated from Fig. 3 A. The effect of increasing the blocking divalent concentration by 50% (to emulate the 4–6-mM change used in our measurements) shown in B demonstrates that a 33% decrease in current amplitude is predicted. This simulation used high affinity block with a [B]/K_D ratio of ∼30 to achieve the relatively flat plateau in the current amplitude over a voltage range of −80 to −140 mV. For comparison, the gating pore currents recorded with a variety of divalents is reproduced from Fig. 2 B. The solid black line represents the model response in 4 mM of bathing divalent, whereas the solid red line shows the predicted current amplitude if the divalent blocker concentration were increased to 6 mM. In contrast, no decrease in current amplitude consistent with this model was observed under any of the conditions in which 6 mM of divalent cations (Ba²⁺, Ca²⁺, or Zn²⁺) were included in the bath. The prediction that increasing the divalent concentration by 50% will produce a substantial reduction of current amplitude (∼30%) is relatively independent of the model assumption for the K_D, provided [B] ≥ K_D as shown in Fig. 5.