Connexin channels and phospholipids: association and modulation

<p>Abstract</p> <p>Background</p> <p>For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood.</p> <p>Results</p> <p>Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred.</p> <p>Conclusion</p> <p>This study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents.</p

Clonally related visual cortical neurons show similar stimulus feature selectivity

A fundamental feature of the mammalian neocortex is its columnar organization(1). In the visual cortex, functional columns consisting of neurons with similar orientation preference have been characterized extensively(2-4), but how these columns are constructed during development remains unclear(5). The ‘radial unit hypothesis’(6) posits that the ontogenetic columns formed by clonally related neurons migrating along the same radial glial fiber during corticogenesis(7) provide the basis for functional columns in adult neocortex(1). However, direct correspondence between the ontogenetic and functional columns has not been demonstrated(8). Here we show that, despite the lack of discernible orientation map in mouse visual cortex(4,9,10), sister neurons in the same radial clone exhibit similar orientation preference. Using a retroviral vector encoding green fluorescent protein (GFP) to label radial clones of excitatory neurons and in vivo two-photon calcium imaging to measure the neuronal response properties, we found that sister neurons preferred similar orientations, while nearby non-sisters showed no such relationship. Interestingly, disruption of gap junction coupling by viral expression of a dominant-negative mutant of Cx26 or by daily administration of a gap junction blocker carbenoxolone (CBX) during the first postnatal week greatly diminished the functional similarity between sister neurons, suggesting that the maturation of ontogenetic into functional columns requires intercellular communication through gap junctions. Together with the recent finding of preferential excitatory connections among sister neurons(11), our results support the radial unit hypothesis and unify the ontogenetic and functional columns in the visual cortex