Electrostatic interactions in the channel cavity as an important determinant of potassium channel selectivity

Abstract

Potassium channels are membrane proteins that allow the passage of potassium ions at near diffusion rates while severely limiting the flux of the slightly smaller sodium ions. Although studies thus far have focused on the narrowest part of the channel, known as the selectivity filter, channels are long pores with multiple ions that traverse the selectivity filter, the water-filled central cavity, and the rest of the pore formed by cytoplasmic domains. Here, we present experimental analyses on Kir3.2 (GIRK2), a G protein-activated inwardly rectifying potassium (Kir) channel, showing that a negative charge introduced at a pore-facing position in the cavity (N184) below the selectivity filter restores both K(+) selectivity and inward rectification properties to the nonselective S177W mutant channel. Molecular modeling demonstrates that the negative residue has no effect on the geometry of the selectivity filter, suggesting that it has a local effect on the cavity ion. Moreover, restoration of selectivity does not depend on the exact location of the charge in the central cavity as long as this residue faces the pore, where it is in close contact with permeant ions. Our results indicate that interactions between permeant ions and the channel cavity can influence ion selectivity and channel block by means of an electrostatic effect.

Fig. 1.

Mutations of the M2 inner transmembrane helix residues control K⁺ selectivity. (A) (Left) Homology model of the Kir3.2 channel with the S177T (blue) mutation on the M2 inner helix, behind the selectivity filter. (Right) Representative current traces obtained from Xenopus oocytes expressing S177T channels bathed in 90 mM Na⁺ or 90 mM K⁺ show that S177T channels carry K⁺ current but not Na⁺ current. (B) Kir3.2 channel with an S177W (red) mutation that fills the space behind the selectivity filter. Current traces illustrate large inward fluxes for both K⁺ and Na⁺ for this nonselective S177W mutant. (C) Kir3.2 double mutant channel with a S177W (red) mutation and a N184D (green) mutation in the center of the central cavity near a K⁺ ion (blue). The double mutant channel has regained K⁺ selectivity and exhibits similar current traces to the K⁺-selective S177T channel. Currents were recorded at membrane potentials ranging from +40 to −150 mV from a holding potential of 0 mV and are shown in 20-mV increments.

Fig. 2.

Average distortions in the selectivity filter introduced by mutant residues. The relative displacement of amino acids from the K⁺ signature sequence, TTIGYG, was deduced from Kir3.2 mutant models. The filters of S177T (blue) and N184D (green dashed) mutant channels are similar to WT models (not shown). However, S177W (red) introduces systematic distortions into the filter (up to 1.5 Å). Consistent with this observation, there is greater variation in the filter geometry for S177W models compared with WT models. The N184D mutant combined with S177W (green) does not restore the pattern of distortion created by S177W (red). The S177W-N184D and N184D distributions are significantly different at all positions as analyzed with nonparametric statistics.

Fig. 3.

Negatively charged amino acids in the cavity restore K⁺ selectivity. Only acidic substitutions at the N184 position of Kir3.2 restore K⁺ selectivity to the nonselective S177W mutant channel. Current traces were elicited with voltage pulses from +40 to −150 (in 10-mV increments) from a holding potential of 0 mV in 90 mM K⁺ (green line) and 90 mM Na⁺ (red line). Only traces at −150 mV are shown. All N184 substitutions are in the background of the S177W mutation. Baseline indicates 0 μA current value. (Scale bars: vertical, 10 μA; horizontal, 20 ms.)

Fig. 4.

Aspartate (D) scan of the central cavity supports an electrostatic mechanism for K⁺ selectivity. (A) Side view of the Kir3.2 channel with the S177W (red) mutation on the M2 helix. In the cavity, three residues from two faces, face 1 (green) and face 2 (red), are shown. Residues that were subjected to Asp (D) substitution are as follows: on face 1, G180, N184, and V188; on face 2, S181, A185, and G189. (B) Asp (D) substitution of residues at every position on face 1 (green), but at none of the positions on face 2 (red), restores K⁺ selectivity, as shown by values of the Na⁺ and K⁺ permeability ratio deduced from the difference in reversal potential measured in 90 mM Na⁺ and 90 mM K⁺ solutions. Values are mean ± SEM; values for selective channels are TPN_Q-corrected. ∗, P < 0.001. (C) Molecular surface representation of the Kir3.2 cavity viewed from the extracellular side. Residues on face 1 (green) are closer to the cavity ion (blue) and have greater solvent exposure than the residues on face 2 (red).

Fig. 5.

Mutations in the M2 helix of Kir3.2 also affect inward rectification properties of S177W-bearing mutant channels. Histograms show the ratio of outward K⁺ current recorded at membrane potential +40 mV (K⁺ out) and the inward K⁺ current recorded at −100 mV (K⁺ in) for all Kir3.2 channels used in this study. (A) Rectification values of the S177W-containing channels with single acidic (D or E) substitutions along the M2 helix. Mutations that restore K⁺ selectivity (in green) also increase inward rectification to values comparable to those of S177T channels (∗, P < 0.01 for N184E; ∗, P < 0.001 for all others), whereas all nonselective K⁺ channels (in red) have impaired rectification. (B) Rectification values for various nonselective mutant channels with positive and neutral substitutions at position N184. Basic substitutions at N184 (N184R/K) have the strongest effect by further reducing rectification. ∗, P < 0.001. Values are reported as mean ± SEM. (C) Representative averaged current–voltage (I–V) relations obtained from Xenopus oocytes expressing Kir3.2 mutant channels. Currents were recorded at membrane potentials ranging from +40 mV to −150 mV in 10-mV increments in 90 mM K⁺ bath solution. Points in the I–V curves represent mean ± SEM for n = 8–10 oocytes.