The effects of lipids on channel function
© BioMed Central Ltd 2009
Published: 6 October 2009
Anionic lipids affect the function of many channels, including connexins, as shown in a recent report in BMC Biology. These effects might follow from direct binding of the anionic lipids to the channels.
Lipids affect channel function
Lipid binding to channels
How are the effects of anionic lipid on channel function to be understood? In some cases the interaction of lipid with a membrane protein is highly specific. For example, a single molecule of the anionic lipid phosphatidylglycerol is seen in the X-ray crystal structure of the heterotrimeric nitrate reductase A, bound in a distinct pocket formed by all three subunits, with three positively charged residues contributing to the binding site . In this case binding is strong as well as structurally specific, because the lipid remains bound during the process of crystallization from detergent solution. However, most lipid molecules interacting with a membrane protein are not buried within the structure in this way, but are located on the transmembrane surface of the protein, forming a ring or annulus around the protein . These annular lipid molecules 'solvate' the transmembrane surface of a membrane protein in the same way that water molecules solvate the surface of a water-soluble protein. Given that the effects of anionic lipid on channel function are generally seen only at high concentrations of the anionic lipid, it is likely that the relevant lipid-protein interactions are relatively weak, and that the observed effects are caused by anionic lipids binding at annular sites on the channel.
Slightly stronger binding of PS than PC at a particular site (K > 1) will ensure that the site is occupied mostly by PS at the relatively high concentrations of anionic lipid required to affect channel function. For example, in a bilayer containing 60% PS, an annular site will be 90% occupied by PS if binding of PS at the site is just five times stronger than binding of PC. This binding would affect channel function if it resulted in a significant conformational change in the channel.
A functionally important annular lipid binding with a preference for anionic lipids has been demonstrated on MscL, for which the 'hot spot' for binding anionic lipid was shown to correspond to a cluster of three positively charged residues ; it is likely that similar clusters of positively charged residues close to the lipid-water interface occur on other membrane proteins. In their recent paper, Locke and Harris  report a mass spectrometric method for detecting specificity in lipid binding to connexin channels. They expressed connexin26 and connexin32 in HeLa cells, isolated hemichannels (that is, channels that had not yet formed gap junctions linking apposed cells) using a non-ionic detergent, and characterized the lipids remaining with the hemichannel preparation; lipids associated with a gap junction preparation were also analyzed. Although all classes of lipid were found associated with the hemichannels, there was an enrichment in anionic lipids, consistent with interaction with clusters of positively charged residues of the type suggested above. Indeed, the crystal structure of connexin26 shows a concentration of positively charged residues at the lipid-water interface, on the intracellular side .
Locke and Harris  also found that lipids with particular combinations of fatty acyl chains remained with the purified connexin26 samples, whereas lipids with other combinations of fatty acyl chains remained with the purified connexin32 samples. It would be surprising if this reflected chain-specific binding to the connexins because the effects of chain structure on lipid binding to membrane proteins are generally small . Another possibility, as described by Locke and Harris , is that the fatty acyl chain compositions of the cells expressing connexin26 and connexion32 are slightly different.
Collective physical properties
Because high concentrations of anionic lipid are needed to affect channel function, there is another possible mechanism for the effects of anionic lipid that needs to be considered. Large changes in the chemical composition of a bilayer will result in changes in collective physical properties of the bilayer, such as its thickness and fluidity, and in the related properties of spontaneous curvature and pressure profile across the membrane ; it is therefore conceivable that any observed changes in channel function follow from changes in these collective physical properties rather than from changes in the pattern of charge and hydrogen-bonding interactions between the lipids and the proteins described above.
What conclusions can be drawn from these studies? It is clear that the presence of anionic lipids, generally at high concentrations, affects the function of a wide range of channels. In MscL the anionic lipids bind to a cluster of positively charged residues close to the lipid-water interface, and similar charge interactions are likely to be important for other channels. It is also likely that the effects of anionic lipids on channel function follow directly from binding to these charge clusters, although the possible importance of the collective physical properties of the lipid bilayer cannot yet be ruled out.
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