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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 cholesterol depletion on Kir2.1–PIP2 interactions.A. Representative Kir2.1 current traces before (red) and after (green) dialyzing neomycin into the cell through a patch pipette. The upper family of traces was recorded in a cell in normal cholesterol conditions and the lower family of traces recorded from a cell depleted of cholesterol by pre-exposure to 5 mM MβCD for 1 h. B: The time courses of Kir2.1 rundown in response (1–100 μM) neomycin. C: The time courses of Kir2.1 rundown in response 10 μM neomycin in control and cholesterol depleted cells. D: The time courses of Kir2.1 rundown in response anti-PIP2 antibodies in control and cholesterol depleted cells (all data show means + SEM, n = 5–10 cells per condition).
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f0005: Impact of cholesterol depletion on Kir2.1–PIP2 interactions.A. Representative Kir2.1 current traces before (red) and after (green) dialyzing neomycin into the cell through a patch pipette. The upper family of traces was recorded in a cell in normal cholesterol conditions and the lower family of traces recorded from a cell depleted of cholesterol by pre-exposure to 5 mM MβCD for 1 h. B: The time courses of Kir2.1 rundown in response (1–100 μM) neomycin. C: The time courses of Kir2.1 rundown in response 10 μM neomycin in control and cholesterol depleted cells. D: The time courses of Kir2.1 rundown in response anti-PIP2 antibodies in control and cholesterol depleted cells (all data show means + SEM, n = 5–10 cells per condition).

Mentions: Earlier studies assessed the functional interactions between Kir channels and PIP2 by over-expressing the channels in Xenopus oocytes and measuring the rundown of the current in excised inside-out macropatches with and without perfusing PIP2 to the cytosolic side of the membrane (eg. [7,8]). These studies showed that Kir currents rapidly decreased after the excision of the patch but could be reactivated by perfusion of PIP2. Furthermore, the rate of recovery depended on the strength of the Kir–PIP2 interactions and could be affected by specific mutations or by using different PIP2 analogs. Thus, measuring PIP2-dependent current rundown or recovery was established as a method to assess the strength of Kir–PIP2 interactions (eg. [7,8]). In our recent study, we used an alternative approach of dialyzing neomycin, an agent known to sequester PIP2 by binding to its headgroups and restricting its ability to interact with proteins [30,31], into the patch pipette in whole-cell configuration [16]. As expected, dialyzing neomycin into the pipette resulted in Kir2.1 rundown expressed in Chinese hamster ovary cells (CHO) [16]. We also showed that cholesterol depletion resulted in a significant delay in the current rundown of Kir2.1. Since a faster rundown is interpreted as a weaker Kir–PIP2 interaction [8], these data led us to hypothesize that cholesterol depletion strengthens Kir2.1–PIP2 interactions [16]. It is important to note that since the currents are normalized to the currents recorded at time 0, a delay in neomycin-induced current rundown cannot be attributed to an increase in local PIP2 concentration. To further characterize this observation, we first show here that neomycin-induced rundown of Kir2.1 current is concentration-dependent. Fig. 1A (upper family of traces) shows typical Kir2.1 whole cell currents in CHO cells at time 0 before the start of the dialysis and 60 s after establishing whole-cell configuration that allows neomycin to diffuse into the cytosol. Fig. 1B shows that the effect is concentration-dependent with 1 μM of neomycin having no effect while concentrations of 10 and 100 μM neomycin induce significant rundown. Also, as was shown in our previous study described above, depletion of the cells of cholesterol using 5 mM MβCD results in a significant delay in the rundown suggesting that cholesterol depletion results in strengthening of Kir2.1–PIP2 interactions (Fig. 1C). To further verify that this effect is specific for PIP2, a complementary series of experiments was performed by dialyzing PIP2 antibodies into the cell. The rundown in this case is significantly slower than with neomycin, which most likely reflects a slower diffusion of the antibodies into the cell but, more importantly, cholesterol depletion has the same effect of slowing the rundown process, which points to strengthening Kir2.1–PIP2 interactions (Fig. 1D). This experiment also excludes the possibilities that the observed delay in the current rundown can be due to the effect of cholesterol on the neomycin access to the membrane or PIP2-independent effects of neomycin on channel function. The possibility that the effect of MβCD on neomycin-induced current rundown can be independent of cholesterol depletion was excluded in our previous study by comparing MβCD and MβCD saturated with cholesterol [16].


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

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

Impact of cholesterol depletion on Kir2.1–PIP2 interactions.A. Representative Kir2.1 current traces before (red) and after (green) dialyzing neomycin into the cell through a patch pipette. The upper family of traces was recorded in a cell in normal cholesterol conditions and the lower family of traces recorded from a cell depleted of cholesterol by pre-exposure to 5 mM MβCD for 1 h. B: The time courses of Kir2.1 rundown in response (1–100 μM) neomycin. C: The time courses of Kir2.1 rundown in response 10 μM neomycin in control and cholesterol depleted cells. D: The time courses of Kir2.1 rundown in response anti-PIP2 antibodies 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

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getmorefigures.php?uid=PMC4232564&req=5

f0005: Impact of cholesterol depletion on Kir2.1–PIP2 interactions.A. Representative Kir2.1 current traces before (red) and after (green) dialyzing neomycin into the cell through a patch pipette. The upper family of traces was recorded in a cell in normal cholesterol conditions and the lower family of traces recorded from a cell depleted of cholesterol by pre-exposure to 5 mM MβCD for 1 h. B: The time courses of Kir2.1 rundown in response (1–100 μM) neomycin. C: The time courses of Kir2.1 rundown in response 10 μM neomycin in control and cholesterol depleted cells. D: The time courses of Kir2.1 rundown in response anti-PIP2 antibodies in control and cholesterol depleted cells (all data show means + SEM, n = 5–10 cells per condition).
Mentions: Earlier studies assessed the functional interactions between Kir channels and PIP2 by over-expressing the channels in Xenopus oocytes and measuring the rundown of the current in excised inside-out macropatches with and without perfusing PIP2 to the cytosolic side of the membrane (eg. [7,8]). These studies showed that Kir currents rapidly decreased after the excision of the patch but could be reactivated by perfusion of PIP2. Furthermore, the rate of recovery depended on the strength of the Kir–PIP2 interactions and could be affected by specific mutations or by using different PIP2 analogs. Thus, measuring PIP2-dependent current rundown or recovery was established as a method to assess the strength of Kir–PIP2 interactions (eg. [7,8]). In our recent study, we used an alternative approach of dialyzing neomycin, an agent known to sequester PIP2 by binding to its headgroups and restricting its ability to interact with proteins [30,31], into the patch pipette in whole-cell configuration [16]. As expected, dialyzing neomycin into the pipette resulted in Kir2.1 rundown expressed in Chinese hamster ovary cells (CHO) [16]. We also showed that cholesterol depletion resulted in a significant delay in the current rundown of Kir2.1. Since a faster rundown is interpreted as a weaker Kir–PIP2 interaction [8], these data led us to hypothesize that cholesterol depletion strengthens Kir2.1–PIP2 interactions [16]. It is important to note that since the currents are normalized to the currents recorded at time 0, a delay in neomycin-induced current rundown cannot be attributed to an increase in local PIP2 concentration. To further characterize this observation, we first show here that neomycin-induced rundown of Kir2.1 current is concentration-dependent. Fig. 1A (upper family of traces) shows typical Kir2.1 whole cell currents in CHO cells at time 0 before the start of the dialysis and 60 s after establishing whole-cell configuration that allows neomycin to diffuse into the cytosol. Fig. 1B shows that the effect is concentration-dependent with 1 μM of neomycin having no effect while concentrations of 10 and 100 μM neomycin induce significant rundown. Also, as was shown in our previous study described above, depletion of the cells of cholesterol using 5 mM MβCD results in a significant delay in the rundown suggesting that cholesterol depletion results in strengthening of Kir2.1–PIP2 interactions (Fig. 1C). To further verify that this effect is specific for PIP2, a complementary series of experiments was performed by dialyzing PIP2 antibodies into the cell. The rundown in this case is significantly slower than with neomycin, which most likely reflects a slower diffusion of the antibodies into the cell but, more importantly, cholesterol depletion has the same effect of slowing the rundown process, which points to strengthening Kir2.1–PIP2 interactions (Fig. 1D). This experiment also excludes the possibilities that the observed delay in the current rundown can be due to the effect of cholesterol on the neomycin access to the membrane or PIP2-independent effects of neomycin on channel function. The possibility that the effect of MβCD on neomycin-induced current rundown can be independent of cholesterol depletion was excluded in our previous study by comparing MβCD and MβCD saturated with cholesterol [16].

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