Interaction of local anesthetics with the K (+) channel pore domain: KcsA as a model for drug-dependent tetramer stability.

Local anesthetics and related drugs block ionic currents of Na (+) , K (+) and Ca ( 2+) conducted across the cell membrane by voltage-dependent ion channels. Many of these drugs bind in the permeation pathway, occlude the pore and stop ion movement. However channel-blocking drugs have also been associated with decreased membrane stability of certain tetrameric K (+) channels, similar to the destabilization of channel function observed at low extracellular K (+) concentration. Such drug-dependent stability may result from electrostatic repulsion of K (+) from the selectivity filter by a cationic drug molecule bound in the central cavity of the channel. In this study we used the pore domain of the KcsA K (+) channel protein to test this hypothesis experimentally with a biochemical assay of tetramer stability and theoretically by computational simulation of local anesthetic docking to the central cavity. We find that two common local anesthetics, lidocaine and tetracaine, promote thermal dissociation of the KcsA tetramer in a K (+) -dependent fashion. Docking simulations of these drugs with open, open-inactivated and closed crystal structures of KcsA yield many energetically favorable drug-channel complexes characterized by nonbonded attraction to pore-lining residues and electrostatic repulsion of K (+) . The results suggest that binding of cationic drugs to the inner cavity can reduce tetramer stability of K (+) channels.

Conditional knockout of protein O-mannosyltransferase 2 reveals tissue-specific roles of O-mannosyl glycosylation in brain development.

The meninges produce essential signaling molecules and major protein components of the pial basement membrane during normal brain development. Disruptions in the pial basement membrane underlie neural ectopia seen in those congenital muscular dystrophies (CMDs) caused by mutations in genes involved in O-mannosyl glycosylation. In mammals, biosynthesis of O-mannosyl glycans is initiated by a complex of mutually indispensable protein O-mannosyltransferases 1 and 2 (POMT1 and 2). To study the roles of O-mannosylation in brain development we generated a conditional allele of POMT2. POMT2 nulllizygosity resulted in embryonic lethality because of a defective Reichert's membrane. Brain-specific deletion of POMT2 resulted in hypoglycosylation of α-dystroglycan (DG) and abolished laminin binding activity. The effect of POMT2 deletion on brain development was dependent on timing, as earlier deletion resulted in more severe phenotypes. Multiple brain malformations including overmigration of neocortical neurons and migration failure of granule cells in the cerebellum were observed. Immunofluorescence staining and transmission electron microscopy revealed that these migration defects were closely associated with disruptions in the pial basement membrane. Interestingly, POMT2 deletion in the meninges (and blood vessels) did not disrupt the development of the neocortex. Thus, normal brain development requires protein O-mannosylation activity in neural tissue but not the meninges. These results suggest that gene therapy should be directed to the neural tissue instead of the meninges.