Numerous studies demonstrated that membrane cholesterol is a major regulator of ion channel function. The goal of this review is to discuss significant advances that have been recently achieved in elucidating the mechanisms responsible for cholesterol regulation of ion channels. The first major insight that comes from growing number of studies that based on the sterol specificity of cholesterol effects, show that several types of ion channels (nAChR, Kir, BK, TRPV) are regulated by specific sterol-protein interactions. This conclusion is supported by demonstrating direct saturable binding of cholesterol to a bacterial Kir channel. The second major advance in the field is the identification of putative cholesterol binding sites in several types of ion channels. These include sites at locations associated with the well-known cholesterol binding motif CRAC and its reversed form CARC in nAChR, BK, and TRPV, as well as novel cholesterol binding regions in Kir channels. Notably, in the majority of these channels, cholesterol is suggested to interact mainly with hydrophobic residues in non-annular regions of the channels being embedded in between transmembrane protein helices. We also discuss how identification of putative cholesterol binding sites is an essential step to understand the mechanistic basis of cholesterol-induced channel regulation. Clearly, however, these are only the first few steps in obtaining a general understanding of cholesterol-ion channels interactions and their roles in cellular and organ functions.

Inwardly rectifying potassium (Kir) channels play an important role in setting the resting membrane potential and modulating membrane excitability. We have recently shown that cholesterol regulates representative members of the Kir family and that in the majority of the cases, cholesterol suppresses channel function. Furthermore, recent data indicate that cholesterol regulates Kir channels by specific sterol-protein interactions, yet the location of the cholesterol binding site in Kir channels is unknown. Using a combined computational-experimental approach, we show that cholesterol may bind to two nonanular hydrophobic regions in the transmembrane domain of Kir2.1 located between adjacent subunits of the channel. The location of the binding regions suggests that cholesterol modulates channel function by affecting the hinging motion at the center of the pore-lining transmembrane helix that underlies channel gating either directly or through the interface between the N and C termini of the channel.

A variety of ion channels are regulated by cholesterol, a major lipid component of the plasma membrane whose excess is associated with multiple pathological conditions. However, the mechanism underlying cholesterol sensitivity of ion channels is unknown. We have recently shown that an increase in membrane cholesterol suppresses inwardly rectifying K ⁺ (Kir2) channels that are responsible for maintaining membrane potential in a variety of cell types. Here we show that cholesterol sensitivity of Kir2 channels depends on a specific region of the C terminus of the cytosolic domain of the channel, the CD loop. Within this loop, the L222I mutation in Kir2.1 abrogates the sensitivity of the channel to cholesterol whereas a reverse mutation in the corresponding position in Kir2.3, I214L, has the opposite effect, increasing cholesterol sensitivity. Furthermore, the L222I mutation has a dominant negative effect on cholesterol sensitivity of Kir2.1 WT. Mutations of 2 additional residues in the CD loop in Kir2.1, N216D and K219Q, partially affect the sensitivity of the channel to cholesterol. Yet, whereas these mutations have been shown to affect activation of the channel by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI( 4 , 5 )P ₂ ], other mutations outside the CD loop that have been previously shown to affect activation of the channel by PI( 4 , 5 )P ₂ had no effect on cholesterol sensitivity. Mutations of the lipid-facing residues of the outer transmembrane helix also had no effect. These findings provide insights into the structural determinants of the sensitivity of Kir2 channels to cholesterol, and introduce the critical role of the cytosolic domain in cholesterol dependent channel regulation.

Voltage-gated K ⁺ (Kv) channels couple the movement of a voltage sensor to the channel gate(s) via a helical intracellular region, the S4–S5 linker. A number of studies link voltage sensitivity to interactions of S4 charges with membrane phospholipids in the outer leaflet of the bilayer. Although the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP ₂ ) in the inner membrane leaflet has emerged as a universal activator of ion channels, no such role has been established for mammalian Kv channels. Here we show that PIP ₂ depletion induced two kinetically distinct effects on Kv channels: an increase in voltage sensitivity and a concomitant decrease in current amplitude. These effects are reversible, exhibiting distinct molecular determinants and sensitivities to PIP ₂ . Gating current measurements revealed that PIP ₂ constrains the movement of the sensor through interactions with the S4–S5 linker. Thus, PIP ₂ controls both the movement of the voltage sensor and the stability of the open pore through interactions with the linker that connects them.