Enrichment of Mammalian Tissues and &lt;em&gt;Xenopus&lt;/em&gt; Oocytes with Cholesterol

Slayden, Alexandria V.; Bisen, Shivantika; Dopico, Alex M.; Bukiya, Anna N.; Rosenhouse‐Dantsker, Avia

doi:10.3791/60734

Cited by 11 publications

(11 citation statements)

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“…Consistent with a recent report from our group ( 45 ), a time course of MCA in vitro enrichment with CLR showed a statistically significant increase in MCA CLR levels within 5 min of MCA incubation in CLR-enriching solution ( Fig. S2 ).…”

Section: Resultssupporting

confidence: 92%

“…CLR enrichment of the biological specimens. For CLR enrichment, we followed a previously described methodology (45). Methyl-beta-cyclodextrin (MβCD) at 5:0.625 mM steroid complex ratio (8:1 molar ratio) was prepared in phosphate buffered saline (PBS) and an electrophysiology bath solution for arteries and myocytes/membrane patches, respectively.…”

Section: J O U R N a L P R E -P R O O Fmentioning

confidence: 99%

“…Methyl-beta-cyclodextrin (MβCD) at 5:0.625 mM steroid complex ratio (8:1 molar ratio) was prepared in phosphate buffered saline (PBS) and an electrophysiology bath solution for arteries and myocytes/membrane patches, respectively. To ensure MβCD saturation with CLR, the solution was shaken at 37°C overnight and filtered prior to its application to specimens (45).…”

Section: J O U R N a L P R E -P R O O Fmentioning

confidence: 99%

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Cholesterol activates BK channels by increasing KCNMB1 protein levels in the plasmalemma

Bukiya

Leo

Jaggar

et al. 2021

Journal of Biological Chemistry

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Section: Resultssupporting

confidence: 92%

Section: J O U R N a L P R E -P R O O Fmentioning

confidence: 99%

Section: J O U R N a L P R E -P R O O Fmentioning

confidence: 99%

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Cholesterol activates BK channels by increasing KCNMB1 protein levels in the plasmalemma

Bukiya

Leo

Jaggar

et al. 2021

Journal of Biological Chemistry

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“…The CLR-Ca 2+ i -slo1 interaction reported here occurs at (a) CLR molar fractions found in native membranes (van Meer et al, 2008;Slayden et al, 2020;Jian and Levitan, in press) and to modulate BK (Dopico et al, 2012;Bukiya and Dopico, 2019), and (b) at physiological Ca 2+ i that gates BK channels: in cerebrovascular myocytes, the activity of sarcoplasmic ryanodine receptors generates local Ca 2+ sparks which, given the close vicinity of membrane BK channels and sarcoplasmic ryanodine receptors in these cells, raises Ca 2+ to 4-30 µM in the vicinity of the BK channel's Ca 2+ -sensors (Pérez et al, 2001). The final effect of CLR on BK channel function, however, is drastically modified by the presence of regulatory β 1 subunits, which are highly abundant in cerebrovascular smooth muscle (Kuntamallappanavar et al, 2017;North et al, 2018): CLR enrichment of middle cerebral arteries leads to a trafficking-dependent increase in membrane levels of BK β 1 , a process that overrides CLR direct inhibition of slo1 channels and leads to an actual increased in BK currents (Bukiya et al, 2021).…”

Section: Dmentioning

confidence: 87%

Cholesterol Inhibition of Slo1 Channels Is Calcium-Dependent and Can Be Mediated by Either High-Affinity Calcium-Sensing Site in the Slo1 Cytosolic Tail

et al. 2021

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Ca 2+ -/voltage-gated K + channels of large conductance (BK) are expressed in the cell membranes of all excitable tissues. Currents mediated by BK channel-forming slo1 homotetramers are consistently inhibited by increases in membrane cholesterol (CLR). The molecular mechanisms leading to this CLR action, however, remain unknown. Slo1 channels are activated by increases in Ca 2+ nearby Ca 2+ -recognition sites in the slo1 cytosolic tail: one high-affinity and one low-affinity sites locate to the Regulator of Conductance for K + (RCK) 1 domain, while another high-affinity site locates within the RCK2 domain. Here we first evaluated the cross-talking between Ca 2+ and CLR on the function of slo1 (cbv1 isoform) channels reconstituted into planar lipid bilayers. CLR robustly reduced channel open probability while barely decreasing unitary current amplitude, with CLR maximal effects being observed at 10-30 µM internal Ca 2+ . CLR actions were not only modulated by internal Ca 2+ levels but also disappeared in absence of this divalent. Moreover, in absence of Ca 2+ , BK channel-activating concentrations of Mg 2+ (10 mM) did not support CLR action. Next, we evaluated CLR actions on channels where the different Ca 2+ -sensing sites present in the slo1 cytosolic domain became nonfunctional via mutagenesis. CLR still reduced the activity of low-affinity Ca 2+ (RCK1:E379A, E404A) mutants. In contrast, CLR became inefficacious when both high-affinity Ca 2+ sites were mutated (RCK1:D367A,D372A, and RCK2:D899N,D900N,D901N,D902N,D903N), yet still was able to decrease the activity of each high-affinity site mutant. Therefore, BK channel inhibition by CLR selectively requires optimal levels of Ca 2+ being recognized by either of the slo1 highaffinity Ca 2+ -sensing sites.

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“…Treatment of Xenopus oocytes with a mixture of cholesterol and lipids has been shown to increase the cholesterol/phospholipid molar ratio of the plasma membrane of oocytes ( 45 , 46 ). Thus, in order to enrich the oocytes with cholesterol we used a 1:1:1 (wt/wt/w) mixture containing cholesterol, porcine brain l-α-phosphatidylethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (Avanti Polar Lipids).…”

Section: Methodsmentioning

confidence: 99%

A molecular switch controls the impact of cholesterol on a Kir channel

Corradi

Bukiya

Miranda

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Significance Cholesterol is one of the main components found in plasma membranes and is involved in lipid-dependent signaling enabled by integral membrane proteins such as inwardly rectifying potassium (Kir) channels. Similar to other ion channels, most of the Kir channels are down-regulated by cholesterol. One of the very few notable exceptions is Kir3.4, which is up-regulated by this important lipid. Here, we discovered and characterized a molecular switch that controls the impact (up-regulation vs. down-regulation) of cholesterol on Kir3.4. Our results provide a detailed molecular mechanism of tunable cholesterol regulation of a potassium channel.

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Enrichment of Mammalian Tissues and <em>Xenopus</em> Oocytes with Cholesterol

Cited by 11 publications

References 0 publications

Cholesterol activates BK channels by increasing KCNMB1 protein levels in the plasmalemma

Cholesterol activates BK channels by increasing KCNMB1 protein levels in the plasmalemma

Cholesterol Inhibition of Slo1 Channels Is Calcium-Dependent and Can Be Mediated by Either High-Affinity Calcium-Sensing Site in the Slo1 Cytosolic Tail

A molecular switch controls the impact of cholesterol on a Kir channel

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