Abstract
The crystal structure of the sensorless pore module of a voltage-gated K(+) (Kv) channel showed that lipids occupy a crevice between subunits. We asked if individual lipid monolayers of the bilayer embody independent modules linked to channel gating modulation. Functional studies using single channel current recordings of the sensorless pore module reconstituted in symmetric and asymmetric lipid bilayers allowed us to establish the deterministic role of lipid headgroup on gating. We discovered that individual monolayers with headgroups that coat the bilayer-aqueous interface with hydroxyls stabilize the channel open conformation. The hydroxyl need not be at a terminal position and the effect is not dependent on the presence of phosphate or net charge on the lipid headgroup. Asymmetric lipid bilayers allowed us to determine that phosphoglycerides with glycerol or inositol on the extracellular facing monolayer stabilize the open conformation of the channel. This indirect effect is attributed to a change in water structure at the membrane interface. By contrast, inclusion of the positively charged lysyl-dioleoyl-phosphatidylglycerol exclusively on the cytoplasmic facing monolayer of the bilayer increases drastically the probability of finding the channel open. Such modulation is mediated by a π-cation interaction between Phe-19 of the pore module and the lysyl moiety anchored to the phosphatidylglycerol headgroup. The new findings imply that the specific chemistry of the lipid headgroup and its selective location in either monolayer of the bilayer dictate the stability of the open conformation of a Kv pore module in the absence of voltage-sensing modules.
Highlights
Lipid bilayer composition and asymmetry modulate Kϩ channel function by unknown mechanisms
The Glycerol Moiety on the Phosphoglyceride Headgroup, and Not the Charge, Dictates the Channel Open Probability—To investigate the functional consequence of an interaction between the pore module (PM) of a Kv channel and the surrounding lipids, PM function was screened in lipid bilayers with varying headgroup chemistry and acyl chains (Fig. 1)
The sensorless PM of KvLm was reconstituted in droplet interface bilayers in which both cytoplasmic and extracellular leaflets were of equal composition; channel function was assayed by monitoring the single channel steady-state activity at a depolarizing potential of 100 mV while bathing both sides of the membrane in 0.5 M KCl
Summary
Lipid bilayer composition and asymmetry modulate Kϩ channel function by unknown mechanisms. Asymmetric lipid bilayers allowed us to determine that phosphoglycerides with glycerol or inositol on the extracellular facing monolayer stabilize the open conformation of the channel This indirect effect is attributed to a change in water structure at the membrane interface. We discovered that the inclusion of lipids with headgroups that coat the extracellular membrane-solution interface with hydroxyl groups such as those found in glycerol, phosphoglycerol, phosphoinositol, and lysyl-phosphoglycerol increased drastically the probability of finding the channel open. This effect was observed to be independent of headgroup charge, number of acyl chains, and whether the chains were methyl branched or not. The implication is that lipids are crucial for proper protein folding [44] and to modulate, in a deterministic manner, the properties of the channel
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