PKC and AMPK regulation of Kv1.5 potassium channels.

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K(+) current (IKur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4-2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems.

A phosphoinositide 3-kinase (PI3K)-serum- and glucocorticoid-inducible kinase 1 (SGK1) pathway promotes Kv7.1 channel surface expression by inhibiting Nedd4-2 protein.

Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition. Altogether, these data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd4-2 is a key regulator of Kv7.1 localization and turnover in epithelial cells.

Protein-induced bilayer perturbations: Lipid ordering and hydrophobic coupling.

The host lipid bilayer is increasingly being recognized as an important non-specific regulator of membrane protein function. Despite considerable progress the interplay between hydrophobic coupling and lipid ordering is still elusive. We use electron spin resonance (ESR) to study the interaction between the model protein gramicidin and lipid bilayers of varying thickness. The free energy of the interaction is up to -6kJ/mol; thus not strongly favored over lipid-lipid interactions. Incorporation of gramicidin results in increased order parameters with increased protein concentration and hydrophobic mismatch. Our findings also show that at high protein:lipid ratios the lipids are motionally restricted but not completely immobilized. Both exchange on and off rate values for the lipid<-->gramicidin interaction are lowest at optimal hydrophobic matching. Hydrophobic mismatch of few A results in up to 10-fold increased exchange rates as compared to the 'optimal' match situation pointing to the regulatory role of hydrophobic coupling in lipid-protein interactions.

Interfacial tryptophan residues: a role for the cation-pi effect?

Integral membrane proteins are characterized by having a preference for aromatic residues, e.g., tryptophan (W), at the interface between the lipid bilayer core and the aqueous phase. The reason for this is not clear, but it seems that the preference is related to a complex interplay between steric and electrostatic forces. The flat rigid paddle-like structure of tryptophan, associated with a quadrupolar moment (aromaticity) arising from the pi-electron cloud of the indole, interacts primarily with moieties in the lipid headgroup region hardly penetrating into the bilayer core. We have studied the interaction between the nitrogen moiety of lipid molecule headgroups and the pi-electron distribution of gramicidin (gA) tryptophan residues (W9, W11, W13, and W15) using molecular dynamics (MD) simulations of gA embedded in two hydrated lipid bilayers composed of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoylphosphatidyl-choline (POPC), respectively. We use a force field model for tryptophan in which polarizability is only implicit, but we believe that classical molecular dynamics force fields are sufficient to capture the most prominent features of the cation-pi interaction. Our criteria for cation-pi interactions are based on distance and angular requirements, and the results from our model suggest that cation-pi interactions are relevant for W(PE)1), W(PE)13, W(PE)15, and, to some extent, W(PC)11 and W(PC)13. In our model, W9 does not seem to engage in cation-pi interactions with lipids, neither in POPE nor POPC. The criteria for the cation-pi effect are satisfied more often in POPE than in POPC, whereas the H-bonding ability between the indole donor and the carbonyl acceptor is similar in POPE and POPC. This suggests an increased affinity for lipids with ethanolamine headgroups to transmembrane proteins enriched in interfacial tryptophans.