Kv channels detect changes in the membrane potential via their voltage-sensing domains (VSDs) that control the status of the S6 bundle crossing (BC) gate. The movement of the VSDs results in a transfer of the S4 gating charges across the cell membrane but only the last 10-20% of the total gating charge movement is associated with BC gate opening, which involves cooperative transition(s) in the subunits. Substituting the proline residue P475 in the S6 of the Shaker channel by a glycine or alanine causes a considerable shift in the voltage-dependence of the cooperative transition(s) of BC gate opening, effectively isolating the late gating charge component from the other gating charge that originates from earlier VSD movements. Interestingly, both mutations also abolished Shaker's sensitivity to 4-aminopyridine, which is a pharmacological tool to isolate the late gating charge component. The alanine substitution (that would promote a α-helical configuration compared to proline) resulted in the largest separation of both gating charge components; therefore, BC gate flexibility appears to be important for enabling the late cooperative step of channel opening.

Voltage-gated K+ (Kv) channels mediate the flow of K+ across the cell membrane by regulating the conductive state of their activation gate (AG). Several Kv channels display slow C-type inactivation, a process whereby their selectivity filter (SF) becomes less or nonconductive. It has been proposed that, in the fast inactivation-removed Shaker-IR channel, the W434F mutation epitomizes the C-type inactivated state because it functionally accelerates this process. By introducing another pore mutation that prevents AG closure, P475D, we found a way to record ionic currents of the Shaker-IR-W434F-P475D mutant at hyperpolarized membrane potentials as the W434F-mutant SF recovers from its inactivated state. This W434F conductive state lost its high K+ over Na+ selectivity, and even NMDG+ can permeate, features not observed in a wild-type SF. This indicates that, at least during recovery from inactivation, the W434F-mutant SF transitions to a widened and noncationic specific conformation.

Alkanols are small aliphatic compounds that inhibit voltage-gated K+ (Kv) channels through a yet unresolved gating mechanism. Kv channels detect changes in the membrane potential with their voltage-sensing domains (VSDs) that reorient and generate a transient gating current. Both 1-Butanol (1-BuOH) and 1-Hexanol (1-HeOH) inhibited the ionic currents of the Shaker Kv channel in a concentration dependent manner with an IC50 value of approximately 50 mM and 3 mM, respectively. Using the non-conducting Shaker-W434F mutant, we found that both alkanols immobilized approximately 10% of the gating charge and accelerated the deactivating gating currents simultaneously with ionic current inhibition. Thus, alkanols prevent the final VSD movement(s) that is associated with channel gate opening. Applying 1-BuOH and 1-HeOH to the Shaker-P475A mutant, in which the final gating transition is isolated from earlier VSD movements, strengthened that neither alkanol affected the early VSD movements. Drug competition experiments showed that alkanols do not share the binding site of 4-aminopyridine, a drug that exerts a similar effect at the gating current level. Thus, alkanols inhibit Shaker-type Kv channels via a unique gating modifying mechanism that stabilizes the channel in its non-conducting activated state.

The marine polycyclic-ether toxin gambierol and 1-butanol (n-alkanol) inhibit Shaker-type Kv channels by interfering with the gating machinery. Competition experiments indicated that both compounds do not share an overlapping binding site but gambierol is able to affect 1-butanol affinity for Shaker through an allosteric effect. Furthermore, the Shaker-P475A mutant, which inverses 1-butanol effect, is inhibited by gambierol with nM affinity. Thus, gambierol and 1-butanol inhibit Shaker-type Kv channels via distinct parts of the gating machinery.

By enhancing the genetic code it is possible to incorporate unnatural amino acids (UAA) with new properties into proteins without restrictions on the site to incorporate the UAA. By using the Amber suppression method in Xenopus oocytes we have obtained high expression levels with the heterologously expressed voltage-gated Shaker potassium channel (Kv) harboring the fluorescent unnatural amino acid Anap at various key regions. Anap is environmentally sensitive which makes it capable of probing local conformational changes in the channel. In contrast to the traditional post-translational fluorophore labeling technique with thiol-chemistry, we are now capable of probing dynamics on the cytosolic side aswell as within the membrane bilayer. This opens a wide field of structural questions to be addressed since the important dynamics usually resides inside the cell or within the membrane. Anap was thus incorporated on each side of the S4 voltage sensor as well as on the S6 cytosolic gate. With voltage-clamp fluorometry we were able to determine that the four voltage sensors activate independently while the pore opening occurs cooperatively. Against previous models, pore opening required two cooperative movements. We also simultaneously labeled both ends of the voltage sensor S4 and found that dynamics of N-and C-termini differ. The successful use of fluorescent UAAs combined with voltage-clamp fluorometry has made it possible to study internal dynamics in electrogenic membrane proteins and will find widespread application in structural biology. 2717-Pos Board B409Offsetting the Electric Field Sensed by K V Channels through Residue Substitutions on Top of S1 Kv channel subunits consist of 6 transmembrane segments (S1-S6) whereby the S1 through S4 segments assemble into a voltage sensing domain (VSD) that detects the membrane electric field. The positively charged S4 segment forms the main component of the VSD and undergoes the largest reorientations upon a membrane de-or hyperpolarization, generating a transient gating current. The S1-S3 segments surround the S4 and facilitate the latter's movement across the hydrophobic plasma-membrane. A positive (lysine) and negative (aspartate) charge substitution scan at the extracellular end of the S1 segment in the Shaker-type Kv1.5 channel indicated that this region is sufficiently close to the S4 segment such that it modulates the local membrane electric field. At positions E268, E272, F273 and E276 a charge substitution or charge introduction exerted a surface charge effect and shifted the voltage dependence of channel opening accordingly. Surprisingly, these residues, which modulated the electric field, did not face the S4 in a predicted 3D structure of the Kv1.5 channel in the open state (homology model based on the crystal structure of the Kv2.1/Kv1.2 chimera). This suggests that the introduced charges affect the electrical field around the S4 segment in the closed state only. In conclusion, residues at the top of the S1 segment can state-dependently offset (polarize) the el...