Relevance of lysine snorkeling in the outer transmembrane domain of small viral potassium ion channels.

Transmembrane domains (TMDs) are often flanked by Lys or Arg because they keep their aliphatic parts in the bilayer and their charged groups in the polar interface. Here we examine the relevance of this so-called "snorkeling" of a cationic amino acid, which is conserved in the outer TMD of small viral K(+) channels. Experimentally, snorkeling activity is not mandatory for Kcv(PBCV-1) because K29 can be replaced by most of the natural amino acids without any corruption of function. Two similar channels, Kcv(ATCV-1) and Kcv(MT325), lack a cytosolic N-terminus, and neutralization of their equivalent cationic amino acids inhibits their function. To understand the variable importance of the cationic amino acids, we reanalyzed molecular dynamics simulations of Kcv(PBCV-1) and N-terminally truncated mutants; the truncated mutants mimic Kcv(ATCV-1) and Kcv(MT325). Structures were analyzed with respect to membrane positioning in relation to the orientation of K29. The results indicate that the architecture of the protein (including the selectivity filter) is only weakly dependent on TMD length and protonation of K29. The penetration depth of Lys in a given protonation state is independent of the TMD architecture, which leads to a distortion of shorter proteins. The data imply that snorkeling can be important for K(+) channels; however, its significance depends on the architecture of the entire TMD. The observation that the most severe N-terminal truncation causes the outer TMD to move toward the cytosolic side suggests that snorkeling becomes more relevant if TMDs are not stabilized in the membrane by other domains.

Salt bridges in the miniature viral channel Kcv are important for function.

The viral potassium channel Kcv comprises only 94 amino acids, which represent the pore module of more complex K(+) channels. As for Kir-type channels, Kcv also has a short N-terminal helix exposed to the cytoplasm, upstream of the first transmembrane domain. Here we show that this helix is relevant for Kcv function. The presence of charged amino acids, which form dynamic inter- and intra-subunit salt bridges is crucial. Electrophysiological measurements, yeast rescue experiments and molecular dynamics simulations show that mutants in which the critical salt bridge formation is impaired have no or reduced channel activity. We conclude that these salt bridges destabilise the complexation of K(+) ions by negative charges on the inner transmembrane domain at the entrance into the cavity. This feature facilitates a continuous and coordinated transfer of ions between the cavity and the cytoplasm for channels without the canonical bundle crossing.

Model development for the viral Kcv potassium channel.

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.

Molecular dynamics simulation of the cytosolic mouth in Kcv-type potassium channels.

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.

Elongation of outer transmembrane domain alters function of miniature K+ channel Kcv.

The virus-coded channel Kcv has the typical structure of a two-transmembrane domain K(+) channel. Exceptional are its cytoplasmic domains: the C terminus basically ends inside the membrane and, hence, precludes the formation of a cytoplasmic gate by the so-called bundle crossing; the cytoplasmic N terminus is composed of only 12 amino acids. According to structural predictions, it is positioned in the membrane/aqueous interface and connected via a proline kink to the outer transmembrane domain (TM1). Here, we show that this proline kink affects channel function by determining the position of TM1 in the membrane bilayer. Extension of the hydrophobic length of TM1 by either eliminating the proline kink or introducing an alanine in TM1 augments a time- and voltage-dependent inward rectification of the channel. This suggests that the positional information of TM1 in the bilayer is transmitted to a channel gate, which is not identical with the cytoplasmic bundle crossing.

Long distance interactions within the potassium channel pore are revealed by molecular diversity of viral proteins.

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 approximately 1 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.

Trafficking of the plant potassium inward rectifier KAT1 in guard cell protoplasts of Vicia faba.

Trafficking of K+ inward (Kin+) rectifying channels was analyzed in guard cells of Vicia faba transfected with the Kin+ rectifier from Arabidopsis thaliana KAT1 fused to the green fluorescent protein (GFP). Confocal images and whole-cell patch-clamp measurements confirmed the incorporation of active KAT1 channels into the plasma membrane of transfected guard cell protoplasts. The Kin+ rectifier 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 Kin+ rectifier in wt and transfected protoplasts. These vesicular membranes revealed a higher channel density than the plasma membrane, suggesting that Kin+ rectifier remains in clusters during trafficking 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.