Limits...
Phosphoinositides decrease ATP sensitivity of the cardiac ATP-sensitive K(+) channel. A molecular probe for the mechanism of ATP-sensitive inhibition.

Fan Z, Makielski JC - J. Gen. Physiol. (1999)

Bottom Line: Biol.Phosphoinositides failed to desensitize adenosine inhibition of K(ATP).These data suggest that (a) phosphoinositides strongly compete with ATP at a binding site residing on Kir6.2; (b) electrostatic interaction is a characteristic property of this competition; and (c) in conjunction with SUR2, phosphoinositides render additional, complex effects on ATP inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tennessee, College of Medicine, Memphis, Tennessee 38163, USA. zfan@physiol.utmem.edu

ABSTRACT
Anionic phospholipids modulate the activity of inwardly rectifying potassium channels (Fan, Z., and J.C. Makielski. 1997. J. Biol. Chem. 272:5388-5395). The effect of phosphoinositides on adenosine triphosphate (ATP) inhibition of ATP-sensitive potassium channel (K(ATP)) currents was investigated using the inside-out patch clamp technique in cardiac myocytes and in COS-1 cells in which the cardiac isoform of the sulfonylurea receptor, SUR2, was coexpressed with the inwardly rectifying channel Kir6.2. Phosphoinositides (1 mg/ml) increased the open probability of K(ATP) in low [ATP] (1 microM) within 30 s. Phosphoinositides desensitized ATP inhibition with a longer onset period (>3 min), activating channels inhibited by ATP (1 mM). Phosphoinositides treatment for 10 min shifted the half-inhibitory [ATP] (K(i)) from 35 microM to 16 mM. At the single-channel level, increased [ATP] caused a shorter mean open time and a longer mean closed time. Phosphoinositides prolonged the mean open time, shortened the mean closed time, and weakened the [ATP] dependence of these parameters resulting in a higher open probability at any given [ATP]. The apparent rate constants for ATP binding were estimated to be 0.8 and 0.02 mM(-1) ms(-1) before and after 5-min treatment with phosphoinositides, which corresponds to a K(i) of 35 microM and 5.8 mM, respectively. Phosphoinositides failed to desensitize adenosine inhibition of K(ATP). In the presence of SUR2, phosphoinositides attenuated MgATP antagonism of ATP inhibition. Kir6.2DeltaC35, a truncated Kir6.2 that functions without SUR2, also exhibited phosphoinositide desensitization of ATP inhibition. These data suggest that (a) phosphoinositides strongly compete with ATP at a binding site residing on Kir6.2; (b) electrostatic interaction is a characteristic property of this competition; and (c) in conjunction with SUR2, phosphoinositides render additional, complex effects on ATP inhibition. We propose a model of the ATP binding site involving positively charged residues on the COOH-terminus of Kir6.2, with which phosphoinositides interact to desensitize ATP inhibition.

Show MeSH

Related in: MedlinePlus

A model to account for phosphatidylinositol desensitization of ATP inhibition of KATP. The ATP molecule is shown as a stick diagram having an ∼120° angle between the adenosine group (ring structures) and the phosphate group (crosshatched end). The protein binding site is depicted as a “hook” with positively charged residues, indicated by ⊕, representing the site for electrostatic interaction with the negatively charged phosphates, and the hook representing a hydrophobic binding site for the adenine moiety. KA denotes the association constant of the step in which a complex of ATP and channel is formed through the binding of the hydrophilic phosphate group, and KB denotes the association constant of the step forming the complex through the binding of the hydrophobic adenosine moiety. These steps are bimolecular bindings presumably with the same binding constants of the individual parts binding to their binding site. KAu and KBu are the association constants of the subsequent binding of the second part of the ATP molecule to the binding site, which is a unimolecular (thus the u in the superscript) process. Binding of PPIs to the charged part of the ATP binding site (denoted in the diagram as negatively charged heads with tails pointing downward) causes a decrease rate of ATP association involving phosphate group (denoted by an apostrophe and smaller arrows). The rightmost state represents the state in which both parts of the ATP molecule are bound to the binding site. The states with adenosine binding to the site (bottom and right) are proposed to cause nonconducting channels.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230641&req=5

Figure 11: A model to account for phosphatidylinositol desensitization of ATP inhibition of KATP. The ATP molecule is shown as a stick diagram having an ∼120° angle between the adenosine group (ring structures) and the phosphate group (crosshatched end). The protein binding site is depicted as a “hook” with positively charged residues, indicated by ⊕, representing the site for electrostatic interaction with the negatively charged phosphates, and the hook representing a hydrophobic binding site for the adenine moiety. KA denotes the association constant of the step in which a complex of ATP and channel is formed through the binding of the hydrophilic phosphate group, and KB denotes the association constant of the step forming the complex through the binding of the hydrophobic adenosine moiety. These steps are bimolecular bindings presumably with the same binding constants of the individual parts binding to their binding site. KAu and KBu are the association constants of the subsequent binding of the second part of the ATP molecule to the binding site, which is a unimolecular (thus the u in the superscript) process. Binding of PPIs to the charged part of the ATP binding site (denoted in the diagram as negatively charged heads with tails pointing downward) causes a decrease rate of ATP association involving phosphate group (denoted by an apostrophe and smaller arrows). The rightmost state represents the state in which both parts of the ATP molecule are bound to the binding site. The states with adenosine binding to the site (bottom and right) are proposed to cause nonconducting channels.

Mentions: To explain the change in the intrinsic ATP binding affinity caused by PPIs, we propose a hypothetical molecular model that provides a mechanistic and structural basis for the ATP and phosphatidylinositol interaction at an ATP binding site. The model is adapted from a model first proposed generically by Jencks 1975. We hypothesize that ATP binding to Kir6.2 occurs through two interactions: an electrostatic interaction between the negatively charged phosphates of ATP with positively charged amino acids of Kir6.2, and a hydrophobic interaction between the nucleotide of ATP and Kir6.2. Fig. 11 depicts the proposed electrostatic interaction (KA and KAu) and hydrophobic interaction (K B and K Bu) where u represents interactions leading to the state where both moieties are bound. The channel is considered closed, probably by a conformational change, when the adenosine moiety and the phosphate group from the same ATP molecule (right configuration) occupy both sites. Occupation of either site lowers the binding energy and favors formation of the double-occupancy configuration. Whether occupation of a single site can also induce the conformational change required to close the channel is not clear, but our experiment using adenosine suggests that binding of the adenosine moiety alone may be sufficient to close the channel, although energetically this is less likely to occur.


Phosphoinositides decrease ATP sensitivity of the cardiac ATP-sensitive K(+) channel. A molecular probe for the mechanism of ATP-sensitive inhibition.

Fan Z, Makielski JC - J. Gen. Physiol. (1999)

A model to account for phosphatidylinositol desensitization of ATP inhibition of KATP. The ATP molecule is shown as a stick diagram having an ∼120° angle between the adenosine group (ring structures) and the phosphate group (crosshatched end). The protein binding site is depicted as a “hook” with positively charged residues, indicated by ⊕, representing the site for electrostatic interaction with the negatively charged phosphates, and the hook representing a hydrophobic binding site for the adenine moiety. KA denotes the association constant of the step in which a complex of ATP and channel is formed through the binding of the hydrophilic phosphate group, and KB denotes the association constant of the step forming the complex through the binding of the hydrophobic adenosine moiety. These steps are bimolecular bindings presumably with the same binding constants of the individual parts binding to their binding site. KAu and KBu are the association constants of the subsequent binding of the second part of the ATP molecule to the binding site, which is a unimolecular (thus the u in the superscript) process. Binding of PPIs to the charged part of the ATP binding site (denoted in the diagram as negatively charged heads with tails pointing downward) causes a decrease rate of ATP association involving phosphate group (denoted by an apostrophe and smaller arrows). The rightmost state represents the state in which both parts of the ATP molecule are bound to the binding site. The states with adenosine binding to the site (bottom and right) are proposed to cause nonconducting channels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230641&req=5

Figure 11: A model to account for phosphatidylinositol desensitization of ATP inhibition of KATP. The ATP molecule is shown as a stick diagram having an ∼120° angle between the adenosine group (ring structures) and the phosphate group (crosshatched end). The protein binding site is depicted as a “hook” with positively charged residues, indicated by ⊕, representing the site for electrostatic interaction with the negatively charged phosphates, and the hook representing a hydrophobic binding site for the adenine moiety. KA denotes the association constant of the step in which a complex of ATP and channel is formed through the binding of the hydrophilic phosphate group, and KB denotes the association constant of the step forming the complex through the binding of the hydrophobic adenosine moiety. These steps are bimolecular bindings presumably with the same binding constants of the individual parts binding to their binding site. KAu and KBu are the association constants of the subsequent binding of the second part of the ATP molecule to the binding site, which is a unimolecular (thus the u in the superscript) process. Binding of PPIs to the charged part of the ATP binding site (denoted in the diagram as negatively charged heads with tails pointing downward) causes a decrease rate of ATP association involving phosphate group (denoted by an apostrophe and smaller arrows). The rightmost state represents the state in which both parts of the ATP molecule are bound to the binding site. The states with adenosine binding to the site (bottom and right) are proposed to cause nonconducting channels.
Mentions: To explain the change in the intrinsic ATP binding affinity caused by PPIs, we propose a hypothetical molecular model that provides a mechanistic and structural basis for the ATP and phosphatidylinositol interaction at an ATP binding site. The model is adapted from a model first proposed generically by Jencks 1975. We hypothesize that ATP binding to Kir6.2 occurs through two interactions: an electrostatic interaction between the negatively charged phosphates of ATP with positively charged amino acids of Kir6.2, and a hydrophobic interaction between the nucleotide of ATP and Kir6.2. Fig. 11 depicts the proposed electrostatic interaction (KA and KAu) and hydrophobic interaction (K B and K Bu) where u represents interactions leading to the state where both moieties are bound. The channel is considered closed, probably by a conformational change, when the adenosine moiety and the phosphate group from the same ATP molecule (right configuration) occupy both sites. Occupation of either site lowers the binding energy and favors formation of the double-occupancy configuration. Whether occupation of a single site can also induce the conformational change required to close the channel is not clear, but our experiment using adenosine suggests that binding of the adenosine moiety alone may be sufficient to close the channel, although energetically this is less likely to occur.

Bottom Line: Biol.Phosphoinositides failed to desensitize adenosine inhibition of K(ATP).These data suggest that (a) phosphoinositides strongly compete with ATP at a binding site residing on Kir6.2; (b) electrostatic interaction is a characteristic property of this competition; and (c) in conjunction with SUR2, phosphoinositides render additional, complex effects on ATP inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Tennessee, College of Medicine, Memphis, Tennessee 38163, USA. zfan@physiol.utmem.edu

ABSTRACT
Anionic phospholipids modulate the activity of inwardly rectifying potassium channels (Fan, Z., and J.C. Makielski. 1997. J. Biol. Chem. 272:5388-5395). The effect of phosphoinositides on adenosine triphosphate (ATP) inhibition of ATP-sensitive potassium channel (K(ATP)) currents was investigated using the inside-out patch clamp technique in cardiac myocytes and in COS-1 cells in which the cardiac isoform of the sulfonylurea receptor, SUR2, was coexpressed with the inwardly rectifying channel Kir6.2. Phosphoinositides (1 mg/ml) increased the open probability of K(ATP) in low [ATP] (1 microM) within 30 s. Phosphoinositides desensitized ATP inhibition with a longer onset period (>3 min), activating channels inhibited by ATP (1 mM). Phosphoinositides treatment for 10 min shifted the half-inhibitory [ATP] (K(i)) from 35 microM to 16 mM. At the single-channel level, increased [ATP] caused a shorter mean open time and a longer mean closed time. Phosphoinositides prolonged the mean open time, shortened the mean closed time, and weakened the [ATP] dependence of these parameters resulting in a higher open probability at any given [ATP]. The apparent rate constants for ATP binding were estimated to be 0.8 and 0.02 mM(-1) ms(-1) before and after 5-min treatment with phosphoinositides, which corresponds to a K(i) of 35 microM and 5.8 mM, respectively. Phosphoinositides failed to desensitize adenosine inhibition of K(ATP). In the presence of SUR2, phosphoinositides attenuated MgATP antagonism of ATP inhibition. Kir6.2DeltaC35, a truncated Kir6.2 that functions without SUR2, also exhibited phosphoinositide desensitization of ATP inhibition. These data suggest that (a) phosphoinositides strongly compete with ATP at a binding site residing on Kir6.2; (b) electrostatic interaction is a characteristic property of this competition; and (c) in conjunction with SUR2, phosphoinositides render additional, complex effects on ATP inhibition. We propose a model of the ATP binding site involving positively charged residues on the COOH-terminus of Kir6.2, with which phosphoinositides interact to desensitize ATP inhibition.

Show MeSH
Related in: MedlinePlus