Action Of Cholesterol Research Articles

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

Calcium- and voltage-gated K+ channels of large conductance (BKs) 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 calcium (Ca2+) nearby Ca2+-recognition sites in the slo1 cytosolic tail: one high-affinity and one low-affinity site locate to the regulator of conductance for K+ (RCK) 1 domain, whereas another high-affinity site locates within the RCK2 domain. Here, we first evaluated the crosstalking between Ca2+ 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 Ca2+ CLR actions were not only modulated by internal Ca2+ levels but also disappeared in absence of this divalent. Moreover, in absence of Ca2+, BK channel-activating concentrations of magnesium (10 mM) did not support CLR action. Next, we evaluated CLR actions on channels where the different Ca2+-sensing sites present in the slo1 cytosolic domain became nonfunctional via mutagenesis. CLR still reduced the activity of low-affinity Ca2+ (RCK1:E379A, E404A) mutants. In contrast, CLR became inefficacious when both high-affinity Ca2+ 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 Ca2+ being recognized by either of the slo1 high-affinity Ca2+-sensing sites. SIGNIFICANCE STATEMENT: Results reveal that inhibition of calcium/voltage-gated K+ channel of large conductance (BK) (slo1) channels by membrane cholesterol requires a physiologically range of internal calcium (Ca2+) and is selectively linked to the two high-affinity Ca2+-sensing sites located in the cytosolic tail domain, which underscores that Ca2+ and cholesterol actions are allosterically coupled to the channel gate. Cholesterol modification of BK channel activity likely contributes to disruption of normal physiology by common health conditions that are triggered by disruption of cholesterol homeostasis.

Biophysical studies of cholesterol effects on chromatin

Changes in chromatin structure regulate gene expression and genome maintenance. Molecules that bind to the nucleosome, the complex of DNA and histone proteins, are key modulators of chromatin structure. Previous work indicated that cholesterol, a ubiquitous cellular lipid, may bind to chromatin in vivo, suggesting a potential function for lipids in modulating chromatin architecture. However, the molecular mechanisms of cholesterol's action on chromatin structure have remained unclear. Here, we explored the biophysical impact of cholesterol on nucleosome and chromatin fibers reconstituted in vitro and characterized in silico the cholesterol binding to the nucleosome. Our findings support that cholesterol assists 10 and 30 nm chromatin formation and induces folding of long chromatin fibers as a result of direct interaction of the cholesterol to six nucleosomal binding sites.

Is cholesterol good or bad for your brain?--NMDARs have a say.

Is cholesterol good or bad for your brain?--NMDARs have a say.

The CRAC Motif Nearest to the N-End of Slo1 Cytosolic C-Tail is the Main Determinant of BK Channel Sensitivity to Membrane Cholesterol

Increased membrane cholesterol (CLR) decreases BK channel activity (Po) (reviewed by Levitan et al., 2010). Electrophysiological data from a recent structure-activity study of CLR and several analogs on BK channel-forming slo1 proteins (cbv1) reconstituted into phospholipid bilayers led us to hypothesize that CLR action resulted from CLR selective and direct recognition by a site(s) in cbv1 (Bukiya et al., 2011).We identified ten Cholesterol Recognition/Interacting Amino acid Consensus (CRAC) domains in cbv1: CRACs 1-3 in the transmembrane core (S0-S6), and CRACs 4-10 in the cytosolic C tail (CTD). Bilayer electrophysiology demonstrated that construct trcbv1S6, which lacked CTD, failed to respond to CLR. However, an extended construct that included CRAC4 (trcbv1-CRAC4) was CLR-sensitive, indicating that a major determinant of CLR sensitivity lies in CRAC4 and/or the linker between this motif and S6. We next removed two CRACs at a time: immediately distal to CRAC6 (trcbv1-CRAC6) and to CRAC8 (trcbv1-CRAC8). Trcbv1-CRAC6 and trcbv1-CRAC8 were CLR-sensitive, further supporting the hypothesis that the S6-CRAC4 linker and/or CRAC4 itself is essential for CLR action. However, elimination of CRACs 7-10 or CRACs 9-10 somewhat decreased CLR action. To determine whether CTD CRACs themselves contributed to CLR responses, we introduced progressive Phe substitutions of signature tyrosines in CTD CRACs. The most drastic reduction of CLR action occurred with Y450F in CRAC4. However, cumulative Tyr to Phe substitutions in CRACs 5-10 gradually reduced CLR action. In conclusion: 1) CRAC4 in BK CTD is the main region contributing to CLR action; 2) the signature CRAC residue Y450 is essential for CLR sensitivity; 3) tyrosines in CRACs distal to CRAC4 also contribute to overall CLR sensitivity of BK channels. Support: R01-HL104631;R37-AA011560 (AMD); UTHSC NI Fellowship (AKS).