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Interplay Between Lipid Modulators of Kir2 Channels: Cholesterol and PIP2.

Rosenhouse-Dantsker A, Epshtein Y, Levitan I - Comput Struct Biotechnol J (2014)

Bottom Line: Consistent with a reduction in PIP2 levels, dialysis of neomycin resulted in a decrease in Kir2.1 and Kir2.3 current amplitudes (current rundown), however, this effect was significantly delayed by cholesterol depletion for both types of channels suggesting that cholesterol depletion strengthens the interaction between Kir2 channels and PIP2.Consistent with these observations, there is a significant structural overlap between cytosolic residues that are critical for the sensitivity of Kir2 channels to the two lipid modulators but based on recent studies, there is little or no overlap between cholesterol and PIP2 binding sites.Taken together, these observations suggest that cholesterol and PIP2 regulate the channels through distinct binding sites but that the signals generated by the binding of the two modulators converge.

View Article: PubMed Central - PubMed

Affiliation: Section of Pulmonary, Critical Care, Sleep, and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, United States.

ABSTRACT
We have shown earlier that Kir2 channels are suppressed by the elevation of membrane cholesterol. Moreover, it is also well known that activation of Kir channels is critically dependent on a regulatory phospholipid, phosphatidylinositol-4,5-bisphosphate (PIP2). In this study we examined the cross-talk between cholesterol and PIP2 in the regulation of Kir2 channels. The strength of Kir2-PIP2 interactions was assessed by acute sequestering of PIP2 with neomycin dialyzed into cells through a patch pipette while simultaneously recording whole cell currents. Consistent with a reduction in PIP2 levels, dialysis of neomycin resulted in a decrease in Kir2.1 and Kir2.3 current amplitudes (current rundown), however, this effect was significantly delayed by cholesterol depletion for both types of channels suggesting that cholesterol depletion strengthens the interaction between Kir2 channels and PIP2. Furthermore, mutation of Kir2.1 that renders the channels' cholesterol insensitive abrogated cholesterol depletion-induced delay in the current rundown whereas reverse mutation in Kir2.3 has the opposite effect. These observations provide further support for the functional cross-talk between cholesterol and PIP2 in regulating Kir2 channels. Consistent with these observations, there is a significant structural overlap between cytosolic residues that are critical for the sensitivity of Kir2 channels to the two lipid modulators but based on recent studies, there is little or no overlap between cholesterol and PIP2 binding sites. Taken together, these observations suggest that cholesterol and PIP2 regulate the channels through distinct binding sites but that the signals generated by the binding of the two modulators converge.

No MeSH data available.


Related in: MedlinePlus

Impact of reverse mutations Kir2.1 L222I and Kir2.3 I214L on cholesterol dependence of Kir–PIP2 interactions.A: The time courses of Kir2.1 WT and Kir2.1-L222I rundown in response 10 μM neomycin in control and cholesterol depleted cells. B: The time courses of Kir2.3 WT and Kir2.3-I214L rundown in response 1 μM neomycin in control and cholesterol depleted cells (all data show means + SEM, n = 5–10 cells per condition).
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f0015: Impact of reverse mutations Kir2.1 L222I and Kir2.3 I214L on cholesterol dependence of Kir–PIP2 interactions.A: The time courses of Kir2.1 WT and Kir2.1-L222I rundown in response 10 μM neomycin in control and cholesterol depleted cells. B: The time courses of Kir2.3 WT and Kir2.3-I214L rundown in response 1 μM neomycin in control and cholesterol depleted cells (all data show means + SEM, n = 5–10 cells per condition).

Mentions: Our earlier studies showed that a single residue substitution of leucine at position 222 to isoleucine abrogates the sensitivity of Kir2.1 channels to cholesterol whereas the reverse substitution of an isoleucine at a corresponding position of Kir2.3 to leucine increases cholesterol sensitivity of Kir2.3 channels [16]. Here, we tested the impact of these reverse mutations on neomycin-induced current rundown. Our new data show that L222I substitution abrogates the effect of cholesterol depletion on the kinetics of neomycin-induced Kir2.1 current rundown (Fig. 3A). As was shown in Fig. 1, the rundown of the WT Kir2.1 is strongly delayed by cholesterol depletion (see triangle symbols) but the time courses of L222I Kir2.1 rundown in control and cholesterol depleted cells are identical (circles), indicating that cholesterol depletion has no effect on the strength of the interaction of Kir2.1 L222I mutants with PIP2. A loss of the delay effect is consistent with the loss of cholesterol sensitivity of Kir2.1 L222I described above.


Interplay Between Lipid Modulators of Kir2 Channels: Cholesterol and PIP2.

Rosenhouse-Dantsker A, Epshtein Y, Levitan I - Comput Struct Biotechnol J (2014)

Impact of reverse mutations Kir2.1 L222I and Kir2.3 I214L on cholesterol dependence of Kir–PIP2 interactions.A: The time courses of Kir2.1 WT and Kir2.1-L222I rundown in response 10 μM neomycin in control and cholesterol depleted cells. B: The time courses of Kir2.3 WT and Kir2.3-I214L rundown in response 1 μM neomycin in control and cholesterol depleted cells (all data show means + SEM, n = 5–10 cells per condition).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4232564&req=5

f0015: Impact of reverse mutations Kir2.1 L222I and Kir2.3 I214L on cholesterol dependence of Kir–PIP2 interactions.A: The time courses of Kir2.1 WT and Kir2.1-L222I rundown in response 10 μM neomycin in control and cholesterol depleted cells. B: The time courses of Kir2.3 WT and Kir2.3-I214L rundown in response 1 μM neomycin in control and cholesterol depleted cells (all data show means + SEM, n = 5–10 cells per condition).
Mentions: Our earlier studies showed that a single residue substitution of leucine at position 222 to isoleucine abrogates the sensitivity of Kir2.1 channels to cholesterol whereas the reverse substitution of an isoleucine at a corresponding position of Kir2.3 to leucine increases cholesterol sensitivity of Kir2.3 channels [16]. Here, we tested the impact of these reverse mutations on neomycin-induced current rundown. Our new data show that L222I substitution abrogates the effect of cholesterol depletion on the kinetics of neomycin-induced Kir2.1 current rundown (Fig. 3A). As was shown in Fig. 1, the rundown of the WT Kir2.1 is strongly delayed by cholesterol depletion (see triangle symbols) but the time courses of L222I Kir2.1 rundown in control and cholesterol depleted cells are identical (circles), indicating that cholesterol depletion has no effect on the strength of the interaction of Kir2.1 L222I mutants with PIP2. A loss of the delay effect is consistent with the loss of cholesterol sensitivity of Kir2.1 L222I described above.

Bottom Line: Consistent with a reduction in PIP2 levels, dialysis of neomycin resulted in a decrease in Kir2.1 and Kir2.3 current amplitudes (current rundown), however, this effect was significantly delayed by cholesterol depletion for both types of channels suggesting that cholesterol depletion strengthens the interaction between Kir2 channels and PIP2.Consistent with these observations, there is a significant structural overlap between cytosolic residues that are critical for the sensitivity of Kir2 channels to the two lipid modulators but based on recent studies, there is little or no overlap between cholesterol and PIP2 binding sites.Taken together, these observations suggest that cholesterol and PIP2 regulate the channels through distinct binding sites but that the signals generated by the binding of the two modulators converge.

View Article: PubMed Central - PubMed

Affiliation: Section of Pulmonary, Critical Care, Sleep, and Allergy, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, United States.

ABSTRACT
We have shown earlier that Kir2 channels are suppressed by the elevation of membrane cholesterol. Moreover, it is also well known that activation of Kir channels is critically dependent on a regulatory phospholipid, phosphatidylinositol-4,5-bisphosphate (PIP2). In this study we examined the cross-talk between cholesterol and PIP2 in the regulation of Kir2 channels. The strength of Kir2-PIP2 interactions was assessed by acute sequestering of PIP2 with neomycin dialyzed into cells through a patch pipette while simultaneously recording whole cell currents. Consistent with a reduction in PIP2 levels, dialysis of neomycin resulted in a decrease in Kir2.1 and Kir2.3 current amplitudes (current rundown), however, this effect was significantly delayed by cholesterol depletion for both types of channels suggesting that cholesterol depletion strengthens the interaction between Kir2 channels and PIP2. Furthermore, mutation of Kir2.1 that renders the channels' cholesterol insensitive abrogated cholesterol depletion-induced delay in the current rundown whereas reverse mutation in Kir2.3 has the opposite effect. These observations provide further support for the functional cross-talk between cholesterol and PIP2 in regulating Kir2 channels. Consistent with these observations, there is a significant structural overlap between cytosolic residues that are critical for the sensitivity of Kir2 channels to the two lipid modulators but based on recent studies, there is little or no overlap between cholesterol and PIP2 binding sites. Taken together, these observations suggest that cholesterol and PIP2 regulate the channels through distinct binding sites but that the signals generated by the binding of the two modulators converge.

No MeSH data available.


Related in: MedlinePlus