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Eur Biophys J
2008 Feb 01;372:165-71. doi: 10.1007/s00249-007-0206-7.
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Non-equivalent role of TM2 gating hinges in heteromeric Kir4.1/Kir5.1 potassium channels.
Shang L
,
Tucker SJ
.
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Comparison of the crystal structures of the KcsA and MthK potassium channels suggests that the process of opening a K(+) channel involves pivoted bending of the inner pore-lining helices at a highly conserved glycine residue. This bending motion is proposed to splay the transmembrane domains outwards to widen the gate at the "helix-bundle crossing". However, in the inwardly rectifying (Kir) potassium channel family, the role of this "hinge" residue in the second transmembrane domain (TM2) and that of another putative glycine gating hinge at the base of TM2 remain controversial. We investigated the role of these two positions in heteromeric Kir4.1/Kir5.1 channels, which are unique amongst Kir channels in that both subunits lack a conserved glycine at the upper hinge position. Contrary to the effect seen in other channels, increasing the potential flexibility of TM2 by glycine substitutions at the upper hinge position decreases channel opening. Furthermore, the contribution of the Kir4.1 subunit to this process is dominant compared to Kir5.1, demonstrating a non-equivalent contribution of these two subunits to the gating process. A homology model of heteromeric Kir4.1/Kir5.1 shows that these upper "hinge" residues are in close contact with the base of the pore alpha-helix that supports the selectivity filter. Our results also indicate that the highly conserved glycine at the "lower" gating hinge position is required for tight packing of the TM2 helices at the helix-bundle crossing, rather than acting as a hinge residue.
Fig. 1. a Cartoons of the closed state KcsA (left) and open state MthK (right) structures. The relative positions of the helix-bundle crossing (HBC) and upper glycine hinge (Hinge) in TM2 (yellow) are indicated. b An alignment of TM2 from a range of different mammalian Kir channels with the KcsA, MthK and Shaker sequences. Note that the upper glycine hinge is not conserved in Kir4.1 or Kir5.1, but that the lower glycine “hinge†residue is highly conserved
Fig. 2. a Relative whole-cell currents for glycine mutations at the upper hinge position in different subunits of heteromeric Kir4.1/Kir5.1 channels. Maximum steady-state currents were recorded at −120 mV, n = 12. b Single-channel open probability values (Po) for the same mutations (see “Methods†and also Table 1). *P < 0.005
Fig. 3. Representative single-channel currents for wild-type Kir4.1/Kir5.1, Kir4.1(T154G)/Kir5.1, Kir4.1/Kir5.1(S157G) and Kir4.1(T154G)/Kir5.1(T157G) mutants recorded in the cell-attached mode at −120 mV. No differences are seen in either the amplitude of the current or the “bursting†single-channel behaviour with multiple sub-conductance states. However, the open probabilities (P0) were decreased and correlate with the reduction in whole-cell current (see also Table 1)
Fig. 4. The effect of different amino acid substitutions at the upper hinge residues (T154 in Kir4.1 subunit and S157 in Kir5.1 subunit) on single-channel open-probability (Po) of heteromeric Kir4.1/Kir5.1 channels. WT wild-type Kir4.1/Kir5.1. Currents were measured in the cell-attached mode at −120 mV, n = 6. *P < 0.005
Fig. 5. Effect of glycine substitutions at positions adjacent to the upper hinge position on whole-cell currents of heteromeric Kir4.1/Kir5.1 channels. WT wild-type Kir4.1/Kir5.1. Maximal steady-state currents were recorded at −120 mV, n = 12. *P < 0.005. **P < 0.05
Fig. 6. Homology model of heteromeric Kir4.1/Kir5.1. For clarity, only TM2 and the pore α-helices are shown. Kir4.1 TM2 is shown in yellow, Kir5.1 TM2 in light blue. The upper hinge residues (Kir4.1-T154, Kir5.1-S157) and the putative lower hinge glycines (Kir4.1-G163, Kir5.1-G166) are indicated as vdwspheres in cpk. Enlarged views of the upper hinge area of Kir4.1 showing T154 and of the lower hinge areas are shown on the right hand side
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