Structural properties determining low K⁺ affinity of the selectivity filter in the TWIK1 K⁺ channel.

Canonical K⁺ channels are tetrameric and highly K⁺-selective, whereas two-pore-domain K⁺ (K2P) channels form dimers, but with a similar pore architecture. A two-pore-domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na⁺ and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange-induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K⁺-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K⁺ ions, WT and both variants exhibit an amide-I band at 1680 cm^-1 This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K⁺, a feature very similar to that of the canonical K⁺ channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K⁺ affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm^-1 band upon changes from K⁺ to Rb⁺ or Cs⁺ conditions. High-speed atomic force microscopy disclosed that TWIK1's surface morphology largely does not change in K⁺ and Na⁺ solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful "molecular fingerprints" for assessing the properties of other K⁺ channels.

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