SummaryTraf®cking of K inward (K in ) rectifying channels was analyzed in guard cells of Vicia faba transfected with the K in recti®er from Arabidopsis thaliana KAT1 fused to the green¯uorescent protein (GFP). Confocal images and whole-cell patch-clamp measurements con®rmed the incorporation of active KAT1 channels into the plasma membrane of transfected guard cell protoplasts. The K in recti®er current density of the plasma membrane was much larger in transfected protoplasts than in wild-type (wt) protoplasts. This shows a coupling between K channel synthesis and incorporation of the channel into the plasma membrane. Pressure-driven increase and decrease in surface area led to the incorporation and removal of vesicular membrane carrying active K in recti®er in wt and transfected protoplasts. These vesicular membranes revealed a higher channel density than the plasma membrane, suggesting that K in recti®er remains in clusters during traf®cking to and from the plasma membrane. The observed results can be explained by a model illustrating that vesicles of a pre-plasma membrane pool carry K channels preferentially in clusters during constitutive and pressure-driven exo-and endocytosis.

A computational model for the open state of the short viral Kcv potassium channel was created and tested based on homology modeling and extensive molecular-dynamics simulation in a membrane environment. Particular attention was paid to the structure of the highly flexible N-terminal region and to the protonation state of membrane-exposed lysine residues. Data from various experimental sources, NMR spectroscopy, and electrophysiology, as well as results from three-dimensional reference interaction site model integral equation theory were taken into account to select the most reasonable model among possible variants. The final model exhibits spontaneous ion transitions across the complete pore, with and without application of an external field. The nonequilibrium transport events could be induced reproducibly without abnormally large driving potential and without the need to place ions artificially at certain key positions along the transition path. The transport mechanism through the filter region corresponds to the classic view of single-file motion, which in our case is coupled to frequent exchange of ions between the innermost filter position and the cavity.

The functional effect of mutations near the intracellular mouth of the short viral Kcv potassium channel was studied by molecular dynamics simulations. As a model system we used the analogously mutated and truncated KirBac1.1, a channel with known crystal structure that shares genuine local sequence motifs with Kcv. By a novel simulated annealing methodology for structural averaging, information about the structure and dynamics of the intracellular mouth was extracted and complemented by Poisson-Boltzmann and 3D-RISM (reference interaction site model) integral equation theory for the determination of the K+ free energy surface. Besides the wild-type analogue of Kcv with its experimental reference activity (truncated KirBac1.1), two variants were studied: a deletion mutant where the N-terminus is further truncated by eight amino acids, showing inactivity in the Kcv reference system, and a point mutant where the kink-forming proline at position 13 is substituted by alanine, resulting in hyperactivity. The computations reveal that the change of activity is closely related to a hydrophilic intracellular constriction formed by the C-terminal residues of the monomers. Hyperactivity of the point mutant is correlated with both sterical and electrostatic factors, while inactivity of the deletion mutant is related to a loss of specific salt bridge patterns between the C- and N-terminus at the constriction and to the consequences for ion passage barriers, as revealed by integral equation theory. The cytosolic gate, however, is probably formed by the N-terminal segment up to the proline kink and not by the constriction. The results are compared with design principles found for other channels.

Kcv is a 94-amino acid protein encoded by chlorella virus PBCV-1 that corresponds to the pore module of K ؉ channels. Therefore, Kcv can be a model for studying the protein design of K ؉ channel pores. We analyzed the molecular diversity generated by ϳ1 billion years of evolution on kcv genes isolated from 40 additional chlorella viruses. Because the channel is apparently required for virus replication, the Kcv variants are all functional and contain multiple and dispersed substitutions that represent a repertoire of allowed sets of amino acid substitutions (from 4 to 12 amino acids). Correlations between amino acid substitutions and the new properties displayed by these channels guided site-directed mutations that revealed synergistic amino acid interactions within the protein as well as previously unknown interactions between distant channel domains. The effects of these multiple changes were not predictable from a priori structural knowledge of the channel pore.