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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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Membrane association of Kir6.2 COOH-terminal fragments. (A–C) Fraction of fluorescence in membrane (mean ± SEM) from cells expressing GFP-PH and GFP-tagged COOH-terminal fragments (mean ± SEM, n indicated). In B and C, results are shown from parallel transfections (n indicated, *, P < 0.05).
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fig4: Membrane association of Kir6.2 COOH-terminal fragments. (A–C) Fraction of fluorescence in membrane (mean ± SEM) from cells expressing GFP-PH and GFP-tagged COOH-terminal fragments (mean ± SEM, n indicated). In B and C, results are shown from parallel transfections (n indicated, *, P < 0.05).

Mentions: Each 1 ml fraction of either supernatant or pellet was excited at 467 nm in a fluorometer. Emission spectra (480–600 nm) of buffer alone and of untransfected cells were collected for each experiment. GFP fluorescence spectra were obtained by subtraction of the appropriate spectrum from untransfected samples and normalized to protein density estimated from E480, i.e., adjusted spectra = (raw spectra – buffer spectra) − constant × (control spectra – buffer spectra). The fractional fluoresecence in the membrane (E[memb]/E[memb + Cyt]), reported in Figs. 4 and 5, was calculated from the emission at 518 nm, with excitation at 467 nm.


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)

Membrane association of Kir6.2 COOH-terminal fragments. (A–C) Fraction of fluorescence in membrane (mean ± SEM) from cells expressing GFP-PH and GFP-tagged COOH-terminal fragments (mean ± SEM, n indicated). In B and C, results are shown from parallel transfections (n indicated, *, P < 0.05).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233865&req=5

fig4: Membrane association of Kir6.2 COOH-terminal fragments. (A–C) Fraction of fluorescence in membrane (mean ± SEM) from cells expressing GFP-PH and GFP-tagged COOH-terminal fragments (mean ± SEM, n indicated). In B and C, results are shown from parallel transfections (n indicated, *, P < 0.05).
Mentions: Each 1 ml fraction of either supernatant or pellet was excited at 467 nm in a fluorometer. Emission spectra (480–600 nm) of buffer alone and of untransfected cells were collected for each experiment. GFP fluorescence spectra were obtained by subtraction of the appropriate spectrum from untransfected samples and normalized to protein density estimated from E480, i.e., adjusted spectra = (raw spectra – buffer spectra) − constant × (control spectra – buffer spectra). The fractional fluoresecence in the membrane (E[memb]/E[memb + Cyt]), reported in Figs. 4 and 5, was calculated from the emission at 518 nm, with excitation at 467 nm.

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