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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 - J. Gen. Physiol. (2004)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.

ABSTRACT
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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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.
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fig7: 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.

Mentions: At five positions tested in detail, the potency, voltage dependence, and kinetics of spermine block are essentially unaltered by introduction of cysteines into the rear half of the dimer construct (Figs. 6 and 7). After complete modification with MTSEA+, the current was reduced to a variable extent in each case (129C, 43%; 157C, 24 ± 2%, n = 3; 164C, 53 ± 1%, n = 3; 169C, 50 ± 2%, n = 3; 212C, 59 ± 1%, n = 4). Although current reduction after MTSEA+ application suggests that cysteine modification has occurred, we cannot be sure whether both cysteines in the channel have reacted, or whether reaction with one cysteine somehow inhibits MTSEA+ modification at a second site in the channel. Given that each subunit in the dimer contains a negatively charged aspartate residue at position 160, complete modification with MTSEA+ at two cysteine residues would result in a net charge of −2 in the inner cavity, whereas modification of a single cysteine would result in a net charge of −3. Modification with MTSEA+ substantially altered the rectification properties in each case. While the effects are most likely due to electrostatic interactions between the positively charged ethylamine groups and the blocking polyamine, we cannot yet rule out steric effects of the MTSEA modification. Most importantly, modification altered rectification in very different ways depending on the modification site. As described below, at locations above N160D (L157C and V129C), modification by MTSEA+ greatly decreases spermine sensitivity, generating steady-state rectification intermediate between that of WT and N160D channels (Fig. 6, B and C).


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 - J. Gen. Physiol. (2004)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234007&req=5

fig7: 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.
Mentions: At five positions tested in detail, the potency, voltage dependence, and kinetics of spermine block are essentially unaltered by introduction of cysteines into the rear half of the dimer construct (Figs. 6 and 7). After complete modification with MTSEA+, the current was reduced to a variable extent in each case (129C, 43%; 157C, 24 ± 2%, n = 3; 164C, 53 ± 1%, n = 3; 169C, 50 ± 2%, n = 3; 212C, 59 ± 1%, n = 4). Although current reduction after MTSEA+ application suggests that cysteine modification has occurred, we cannot be sure whether both cysteines in the channel have reacted, or whether reaction with one cysteine somehow inhibits MTSEA+ modification at a second site in the channel. Given that each subunit in the dimer contains a negatively charged aspartate residue at position 160, complete modification with MTSEA+ at two cysteine residues would result in a net charge of −2 in the inner cavity, whereas modification of a single cysteine would result in a net charge of −3. Modification with MTSEA+ substantially altered the rectification properties in each case. While the effects are most likely due to electrostatic interactions between the positively charged ethylamine groups and the blocking polyamine, we cannot yet rule out steric effects of the MTSEA modification. Most importantly, modification altered rectification in very different ways depending on the modification site. As described below, at locations above N160D (L157C and V129C), modification by MTSEA+ greatly decreases spermine sensitivity, generating steady-state rectification intermediate between that of WT and N160D channels (Fig. 6, B and C).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus