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Structural and functional determinants of conserved lipid interaction domains of inward rectifying Kir6.2 channels.

Cukras CA, Jeliazkova I, Nichols CG - J. Gen. Physiol. (2002)

Bottom Line: To further elucidate the mechanistic basis, we examined the membrane association of Kir6.2 fragments of K(ATP) channels, and the effects of site-directed mutations of these fragments and full-length Kir6.2 on membrane association and K(ATP) channel activity, respectively.GFP-tagged Kir6.2 COOH terminus and GFP-tagged pleckstrin homology domain from phospholipase C delta1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic m1 receptor-mediated phospholipid depletion.Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.

View Article: PubMed Central - PubMed

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

ABSTRACT
All members of the inward rectifiier K(+) (Kir) channel family are activated by phosphoinositides and other amphiphilic lipids. To further elucidate the mechanistic basis, we examined the membrane association of Kir6.2 fragments of K(ATP) channels, and the effects of site-directed mutations of these fragments and full-length Kir6.2 on membrane association and K(ATP) channel activity, respectively. GFP-tagged Kir6.2 COOH terminus and GFP-tagged pleckstrin homology domain from phospholipase C delta1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic m1 receptor-mediated phospholipid depletion. Kir COOH termini are predicted to contain multiple beta-strands and a conserved alpha-helix (residues approximately 306-311 in Kir6.2). Systematic mutagenesis of D307-F315 reveals a critical role of E308, I309, W311 and F315, consistent with residues lying on one side of a alpha-helix. Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.

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Dominant-negative inhibition by W311A and F315A mutants. (A) Current density (in zero ATP) in patches expressing Kir6.2[L157C]+SUR1 (L157C), or Kir6.2[L157C]+SUR1 coexpressed with other mutant Kir6.2 subunits, relative to currents in Kir6.2[L157C]+SUR1 patches. Dashed lines indicate predictions assuming that subunits are expressed at equal density, and that at least three K222A subunits are necessary to inhibit current, or only one W311A or F315A subunit is sufficient to inhibit activity. (B) Steady-state dependence of membrane current on [ATP] (relative to current in zero ATP, Irel) for wild type or L157C mutant channels (dashed, from [Shyng et al., 2000]). Superimposed data points are from individual patches in which L157C subunits were coexpressed with K222A (○), W311A ([▪), or F315A(♦) mutant subunits.
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fig3: Dominant-negative inhibition by W311A and F315A mutants. (A) Current density (in zero ATP) in patches expressing Kir6.2[L157C]+SUR1 (L157C), or Kir6.2[L157C]+SUR1 coexpressed with other mutant Kir6.2 subunits, relative to currents in Kir6.2[L157C]+SUR1 patches. Dashed lines indicate predictions assuming that subunits are expressed at equal density, and that at least three K222A subunits are necessary to inhibit current, or only one W311A or F315A subunit is sufficient to inhibit activity. (B) Steady-state dependence of membrane current on [ATP] (relative to current in zero ATP, Irel) for wild type or L157C mutant channels (dashed, from [Shyng et al., 2000]). Superimposed data points are from individual patches in which L157C subunits were coexpressed with K222A (○), W311A ([▪), or F315A(♦) mutant subunits.

Mentions: In a previous systematic mutagenesis of positive charges in the Kir6.2 COOH terminus (Shyng et al., 2000), it was striking that all 22 mutants could produce functional subunits, although in some cases, channel activity was observed only after application of PIP2, or after coexpression with high open state stability mutants such as Kir6.2[L157C], which expresses with high open probability and low ATP sensitivity (Enkvetchakul et al., 2000). Substitution of W311 or F315 with A, R, or D results in no detectable channel activity even after application of 100 μg/ml PIP2 (n = 3–5 patches in each case; unpublished data). Moreover, W311A and F315A mutants are not rescued by coexpression with L157C mutants (Fig. 3 A). W311A and F315A mutants actually cause a strong dominant-negative suppression of L157C subunits (Fig. 3 A). The only detectable currents correspond to those expected of pure L157C+SUR1 currents (Fig. 3 B), consistent with incorporation of even one W311A or F315A subunit into a tetramer being sufficient to abolish activity.


Structural and functional determinants of conserved lipid interaction domains of inward rectifying Kir6.2 channels.

Cukras CA, Jeliazkova I, Nichols CG - J. Gen. Physiol. (2002)

Dominant-negative inhibition by W311A and F315A mutants. (A) Current density (in zero ATP) in patches expressing Kir6.2[L157C]+SUR1 (L157C), or Kir6.2[L157C]+SUR1 coexpressed with other mutant Kir6.2 subunits, relative to currents in Kir6.2[L157C]+SUR1 patches. Dashed lines indicate predictions assuming that subunits are expressed at equal density, and that at least three K222A subunits are necessary to inhibit current, or only one W311A or F315A subunit is sufficient to inhibit activity. (B) Steady-state dependence of membrane current on [ATP] (relative to current in zero ATP, Irel) for wild type or L157C mutant channels (dashed, from [Shyng et al., 2000]). Superimposed data points are from individual patches in which L157C subunits were coexpressed with K222A (○), W311A ([▪), or F315A(♦) mutant subunits.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233865&req=5

fig3: Dominant-negative inhibition by W311A and F315A mutants. (A) Current density (in zero ATP) in patches expressing Kir6.2[L157C]+SUR1 (L157C), or Kir6.2[L157C]+SUR1 coexpressed with other mutant Kir6.2 subunits, relative to currents in Kir6.2[L157C]+SUR1 patches. Dashed lines indicate predictions assuming that subunits are expressed at equal density, and that at least three K222A subunits are necessary to inhibit current, or only one W311A or F315A subunit is sufficient to inhibit activity. (B) Steady-state dependence of membrane current on [ATP] (relative to current in zero ATP, Irel) for wild type or L157C mutant channels (dashed, from [Shyng et al., 2000]). Superimposed data points are from individual patches in which L157C subunits were coexpressed with K222A (○), W311A ([▪), or F315A(♦) mutant subunits.
Mentions: In a previous systematic mutagenesis of positive charges in the Kir6.2 COOH terminus (Shyng et al., 2000), it was striking that all 22 mutants could produce functional subunits, although in some cases, channel activity was observed only after application of PIP2, or after coexpression with high open state stability mutants such as Kir6.2[L157C], which expresses with high open probability and low ATP sensitivity (Enkvetchakul et al., 2000). Substitution of W311 or F315 with A, R, or D results in no detectable channel activity even after application of 100 μg/ml PIP2 (n = 3–5 patches in each case; unpublished data). Moreover, W311A and F315A mutants are not rescued by coexpression with L157C mutants (Fig. 3 A). W311A and F315A mutants actually cause a strong dominant-negative suppression of L157C subunits (Fig. 3 A). The only detectable currents correspond to those expected of pure L157C+SUR1 currents (Fig. 3 B), consistent with incorporation of even one W311A or F315A subunit into a tetramer being sufficient to abolish activity.

Bottom Line: To further elucidate the mechanistic basis, we examined the membrane association of Kir6.2 fragments of K(ATP) channels, and the effects of site-directed mutations of these fragments and full-length Kir6.2 on membrane association and K(ATP) channel activity, respectively.GFP-tagged Kir6.2 COOH terminus and GFP-tagged pleckstrin homology domain from phospholipase C delta1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic m1 receptor-mediated phospholipid depletion.Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.

View Article: PubMed Central - PubMed

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

ABSTRACT
All members of the inward rectifiier K(+) (Kir) channel family are activated by phosphoinositides and other amphiphilic lipids. To further elucidate the mechanistic basis, we examined the membrane association of Kir6.2 fragments of K(ATP) channels, and the effects of site-directed mutations of these fragments and full-length Kir6.2 on membrane association and K(ATP) channel activity, respectively. GFP-tagged Kir6.2 COOH terminus and GFP-tagged pleckstrin homology domain from phospholipase C delta1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic m1 receptor-mediated phospholipid depletion. Kir COOH termini are predicted to contain multiple beta-strands and a conserved alpha-helix (residues approximately 306-311 in Kir6.2). Systematic mutagenesis of D307-F315 reveals a critical role of E308, I309, W311 and F315, consistent with residues lying on one side of a alpha-helix. Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.

Show MeSH
Related in: MedlinePlus