The selectivity filter of a potassium channel, murine kir2.1, investigated using scanning cysteine mutagenesis

Abstract

We have produced a structural model of the pore-forming H5 (or P) region of the strong inward rectifier K+ channel, Kir2.1, based initially on an existing molecular model of the pore region of the voltage-gated K+ channel, Kv1.3. Cysteine-scanning mutagenesis and subsequent blockage by Ag+ was used to test our model by determining the residues in H5 whose side chains line the ion conduction pathway. Mutations made in eight positions within the highly conserved H5 region resulted in apparently non-functional channels. Constructing covalently linked dimers, which carry a cysteine substitution in only one of the linked subunits, rescued six of these mutants; a covalently linked tetramer, carrying a cysteine substitution on only one of the linked subunits, rescued a further mutant. Our results using the dimers and tetramers suggest that residues Thr141, Thr142, Ile143, Tyr145, Phe147 and Cys149 are accessible to externally applied Ag+ (100-200 nM) and therefore that their side chains line the channel pore. We conclude that the topology of the Kir pore is similar, but not identical, to that of Kv channels. Additionally, the molecular model suggests that selectivity may be conferred both by aromatic residues (Tyr145 and Phe147) via cation-pi interactions and by backbone carbonyl groups (Thr142 and Gly144).

Figure 1. Structure of voltage-gated and inwardly rectifying K⁺ channels

A, Kv channel subunits (left) contain 6 membrane-spanning segments (S1-S6) with a short loop of amino acids known as the H5 or P region between S5 and S6. Inward rectifier subunits (right) contain only 2 transmembrane segments but retain H5. B, sequence alignment of the H5 regions of two Kv channels and the strong inward rectifier Kir2.1. The K⁺ channel signature sequence is shown in bold.

Figure 2. Cysteine scan results

A, whole-cell currents recorded from single CHO cells transfected with the dimeric Kir2.1 constructs encoding for cysteine-substituted channels. ‘C149′ indicates a C149S-wild-type dimer. Currents recorded in response to voltage steps from a holding potential of -17 mV (E_K; [K⁺]_o= 70 mm; [K⁺]_i= 140 mm) to test potentials of +33 mV and -102 mV. Control records are indicated by •, currents recorded from the same cell after 90 s exposure to 200 nm external Ag⁺ are indicated by ○. B, histogram showing the percentage inhibition of the whole-cell current for the H5 mutants measured at -102 mV following 90 s exposure to 200 nm external Ag⁺. † Q140C as a tetramer. ² I143C was expressed as a monomer. R indicates the Arg148 mutant, which did not give currents (see text). *P < 0.05.

Figure 3. Rate of Ag⁺ blockage and the depth of the residue

A, fractional remaining current (log scale) for 4 cysteine-substituted mutants all expressed from dimeric constructs plotted against exposure time to 200 nm external Ag⁺. Results are fitted with a single exponential to give a time constant for the rate of decay. Time constants are given in Table 1. B, the relative rate of blockage by Ag⁺ and the distance along the pore axis at which blockage occurs. The distance is measured in the model shown in Fig. 4 from the ring formed by the 4 sulphur atoms of Cys149 lining the pore (i.e. 1 per subunit) to the ring formed by the equivalent 4 atoms in the respective residue.

Figure 4. Molecular model of the H5 region of Kir2.1

A, schematic ‘ball and stick’ representation of the pore region of Kir2.1, as viewed from outside the cell. Residues found to be blocked by Ag⁺ are shown in red (Cys149, Phe147 and Tyr145); residues Thr141 and Ile143 are not visible from this view. The salt bridge between Glu138 and Arg148 in adjacent subunits is indicated in cyan. Residues not blocked by Ag⁺ are shown in blue. The other parts of each subunit are coloured differently from each other. B, space-filling representation of the central pore region viewed from outside the cell. C, as B but viewed from inside the channel looking out. Note that although a methyl group on Ile143 appears to block the pore, this can reorientate so that it moves away from the centre of the pore.