Kurata HT et al. (2004), Molecular basis of inward rectification: polyam...

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J Gen Physiol 2004 Nov 01;1245:541-54. doi: 10.1085/jgp.200409159.

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Molecular basis of inward rectification: polyamine interaction sites located by combined channel and ligand mutagenesis.

Kurata HT , Phillips LR , Rose T , Loussouarn G , Herlitze S , Fritzenschaft H , Enkvetchakul D , Nichols CG , Baukrowitz T .

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Polyamines cause inward rectification of (Kir) K+ channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSEA+ modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter.

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Species referenced: Xenopus laevis
Genes referenced: dtl kcnj11 kcnj2 nbl1 tbx2

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	Figure 1. . The inner cavity of Kir channels. (A) Selected sequence alignment of the pore (P) M2 region and cytoplasmic vestibule-lining region of Kir2.1, Kir6.2, and KirBac1.1. Identical residues in all three sequences are in red. Residues in bold were mutated to glutamates in this study. (B) Structure of Kirbac1.1, illustrating the results of scanning glutamate mutagenesis of Kir6.2 (see data in Fig. 2). Side chains of residues equivalent to those that induce strong spermine rectification, when glutamate is introduced, are indicated in red. Residues equivalent to those that do not affect rectification when glutamate is introduced are indicated in green. Residues are numbered according to Kirbac1.1 (and Kir6.2 in parentheses). Residue 151 (equivalent to Q173 in Kir6.2) is colored orange to indicate the intermediate rectification that is induced when mutated to glutamate.
	Figure 2. . All-or-nothing nature of glutamate-induced rectification in the inner cavity. (A) Currents in response to a voltage ramp from −100 to +100 mV are shown from inside-out membrane patches excised from Xenopus oocytes in the presence and absence of 100 μM spermine. Data from WT and mutant channels, as indicated, are normalized to current at −100 mV. (B) K1/2(0) and zδ for spermine block of WT and mutant channels, obtained from I–V relationships as in A (current in spermine/control current, fitted with single Boltzmann functions). White circles denote WT and mutant channels with zδ < 1 and K1/2(0) > 1 mM. (C) Cartoon illustrating approximate physical location of the residues in the inner cavity and selectivity filter that induce strong rectification.
	Figure 3. . Potency of diamine block of Kir6.2 increases with chain length. (A) Representative currents from voltage step protocols illustrating currents in the absence of any diamine or polyamine (Control), and rectification induced by spermine and diamines of different alkyl chain length in WT and N160E mutant channels. In each case, the scale bar represents 200 ms and 3 nA. Diamines and polyamines were applied at 100 μM, unless otherwise indicated. (B and C) Representative steady-state current–voltage relationships (left), and relative conductance (Grel)–voltage relationships (right). Grel–V curves were fit with single Boltzmann functions, with an offset for nonblocked current (see Fig. 5).
	Figure 4. . Potency of diamine block depends on glutamate location. (A–D) Left, representative currents from voltage step protocols illustrating currents in the absence of diamine/polyamine (Control), and rectification induced by spermine and diamines of different alkyl chain length in glutamate mutant channels (left). Scale bar represents 200 ms and 0.5 nA (A and B) or 2 nA (C and D). Diamines and polyamines were applied at 100 μM, unless otherwise indicated. Right, relative conductance (Grel)–voltage relationships, obtained as in Fig. 3, were fit with single Boltzmann functions, with an offset for nonblocked current (see Fig. 5).
	Figure 5. . Energetic coupling of diamines with introduced glutamates, depends on location. (A–C) Averaged (mean ± SEM, n = 4–8) values of (A) zδ, (B) K1/2(0), and (C) unblocked offset, obtained from fitted Grel–voltage relationships from experiments like those shown in Figs. 3 and 4 for WT and mutant channels, as indicated. For this analysis, blockade of glutamate mutants was examined using diamine concentrations of 10 or 100 μM, and a spermine concentration of 10 or 100 μM. Blockade of WT Kir6.2 channels was examined using diamine concentrations of 100 μM (for DA7 and longer diamines) or 1 mM (for DA6 and shorter diamines, and spermine). (D) Thermodynamic cycle analysis of energetic coupling between diamines of different length and introduced glutamates, relative to the glutamate-less WT channels, using K1/2(0) values from B. Shown are Ω values computed for different length DAn compounds with respect to DA4 (see text).
	Figure 6. . In situ introduction of positive charge to the inner cavity decreases spermine-dependent rectification. (A) Representative currents illustrating rectification induced by spermine in WT-N160D dimer channels. Two voltage protocols are indicated. (B) Representative currents illustrating rectification induced by spermine in WT-N160D, and in V129C, L157C, and L164C mutant channels (mutation in second half of N160D-N160D dimer background), before and after complete modification by MTSEA+ (modification not shown). Voltage protocols as in A. Current scale indicates 0.5 nA in each case. (C) Steady-state current in spermine relative to control (Grel) plotted versus voltage, fitted with Boltzmann functions (no offset) as indicated.
	Figure 7. . In situ introduction of positive charge at the bottom of, or below, the inner cavity slows spermine block kinetics. (A and B) Representative currents illustrating rectification induced by spermine in (A) M169C and (B) S212C mutant channels (mutation in second half of N160D-N160D dimer background), before and after complete modification by MTSEA+ (modification not shown). Current scale indicates 0.2 nA. Note changes of current and time scales, post-modification. (C) Steady-state current in spermine relative to control (Grel) plotted versus voltage, fitted with Boltzmann functions as indicated. (D) Time constant of spermine block/unblock obtained from single exponential fits to current relaxations (from experiments as in A). Block and unblock rates of both M169C and S212C channels are slowed ∼100-fold after modification.
	Figure 8. . Mechanism of polyamine block. (A) Model of the Kir permeation pathway based on KirBac1.1 structure. On the Kir6.2[N160D] (purple) background, certain residues (red and orange) significantly decrease spermine potency when a positive charge is introduced by MTSEA+ modification. Other residues (green) slow the spermine block rate but do not affect steady-state potency. Other critical residues are indicated in blue. (B) We propose that spermine (shown as space-filling model in extended conformation) interacts with cytoplasmic pore residues (left) en route to a blocking site at the top of the inner cavity, extending into the selectivity filter (right). In reaching the deep site, K+ ions from Scav and the selectivity filter are displaced to the external solution, and only the outermost binding site(s) can be occupied by K+, which can electrostatically interact with the blocking spermine. (C) MTSEA+ modification of S212C or M169C (green) slows spermine entry to the deep blocking site, whereas modification of L164 (orange), L157C, or V129C (red) reduces the relative affinity at the deep site. (D) Destabilization of the selectivity filter structure, by disruption of the E126-R136 salt bridge (Yang et al., 1997; Dibb et al., 2003) (blue) removes the barrier to spermine permeation and abolishes rectification.

References [+] :

Brelidze, A ring of eight conserved negatively charged amino acids doubles the conductance of BK channels and prevents inward rectification. 2003, Pubmed, Xenbase