Voltage-activated potassium (KV) channels are potassium selective integral membrane proteins formed by the assembly of four homologous subunits. In response to a membrane depolarization, KV channels open allowing K+ to permeate. In some members of KV channels, sustain depolarization leads to inactivation caused by an N terminus gate. In shaker KV channels, the first 20 amino acids at the NH2 terminus of the protein are essential in enabling it to act as a gate. The tip of the NH2 terminus interacts with residues in the intracellular cavity of KV channels, blocking the permeation of K+. By nature of their tetrameric architecture, inactivating KV channels have four N terminus gates and a set of four sites of action in the intracellular cavity. Yet, N-type inactivation is produced by the binding of only one N terminus gate. Is the site of action in the pore specific to the subunit to which the bound N terminus belongs? To study the interactions between the N terminus gate and the site of action we first constructed a Shaker concatemer KV channel having only one free N terminus gate. The three subunits concatenated at the N terminus have the well-known Δ6-46 inactivation removing deletion. In addition, all subunits contained a mutation to remove C-type inactivation (T449V), which will greatly simplify our kinetic analysis. In this concatemer, we observed a fourfold reduction in the association rate relative to Shaker homotetramers, consistent with the presence of only one N terminus gate relative to four (Figure 1 left, center). In Shaker homotetramers, the tip of the N terminus gate acts on position 470 at the intracellular cavity of the channel. Mutating this residue from isoleucine to valine (I470V) has a dramatic effect on the extent of N-type inactivation. This effect was reproduced in our concatemer by mutating I470V in all subunits. We reasoned that if the N terminus gate interacts with only a single subunit inside the pore to produce inactivation, then a single I470V mutation at this interacting subunit should produce a reduction in inactivation comparable to that observed when all four subunits are mutated to I470V. We are presently testing this hypothesis. We also study the gating mechanisms of transporters, like the Na/K-ATPase. This enzyme, a member of the P-type family (named for their phosphorylated intermediates), harnesses the energy from the hydrolysis of one ATP to alternately export 3Na ions and import 2K ions against their electrochemical gradients. By performing this active transport, the Na/K pump plays an essential role in the homeostasis of intracellular Na and K that is crucial to sustaining cell excitability, volume, and Na-dependent secondary transport. On the basis of biochemical data accumulated during the decade following its discovery, the Na/K-ATPase was proposed to alternately transport Na and K ions according to a model known as the Post-Albers scheme. As ions are transported through the Na/K pump, they become temporarily occluded within the protein, inaccessible from either side, before being released. By restricting Na/K pumps to only the reversible transitions associated with deocclusion and extracellular release of Na+, it is possible to detect pre-steady state electrical signals accompanying those transitions. The signals arise because Na+ traverse a fraction of the membrane potential as they enter or leave their binding sites deep within the pump. At a fixed membrane potential and external sodium concentration, the populations of pumps with empty binding sites, and those with bound or occluded Na, reach a steady-state distribution. A sudden change of membrane voltage then shifts the Na-binding equilibrium, and initiates a redistribution of the pump populations towards a new steady state. The consequent change in Na-binding-site occupancy causes Na to travel between the extracellular environment and the pump interior. In so doing they generate a current. As the system approaches a new steady distribution, fewer Na move, and the current declines. The electrical signals therefore appear as transient currents. Using the squid giant axon preparation, which exploits axial current delivery to generate very fast membrane voltage steps, we previously identified three phases of relaxation in transient pump currents (Holmgren et al., 2000): fast (comparable to the voltage-jump time course), medium-speed (tm 0.2-0.5 ms), and slow (ts 1-10 ms). We suggested that each phase reflects a distinct Na-binding event (or release, depending on the direction of the voltage change) with its associated conformational transition (occlusion or deocclusion). In other words, the Na/K-ATPase undergoes dynamic rearrangements that open external gates to allow bound Na access to the extracellular environment immediately prior to release. We would like to understand how these gates operate, the precise dynamic relationships between the three events that release individual Na+ from the Na/K-ATPase, the thermodynamic principles that govern these conformational changes, the structural movements underlying these events, as well as the type of structural dynamics associated with them.

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Bocksteins, Elke; Snyders, Dirk J; Holmgren, Miguel (2017) Independent movement of the voltage sensors in KV2.1/KV6.4 heterotetramers. Sci Rep 7:41646
Miranda, Pablo; Giraldez, Teresa; Holmgren, Miguel (2016) Interactions of divalent cations with calcium binding sites of BK channels reveal independent motions within the gating ring. Proc Natl Acad Sci U S A 113:14055-14060
Castillo, Juan P; Rui, Huan; Basilio, Daniel et al. (2015) Mechanism of potassium ion uptake by the Na(+)/K(+)-ATPase. Nat Commun 6:7622
Sánchez-Rodríguez, Jorge E; Khalili-Araghi, Fatemeh; Miranda, Pablo et al. (2015) A structural rearrangement of the Na+/K+-ATPase traps ouabain within the external ion permeation pathway. J Mol Biol 427:1335-44
Venkataraman, Gaurav; Srikumar, Deepa; Holmgren, Miguel (2014) Quasi-specific access of the potassium channel inactivation gate. Nat Commun 5:4050
Oelstrom, Kevin; Goldschen-Ohm, Marcel P; Holmgren, Miguel et al. (2014) Evolutionarily conserved intracellular gate of voltage-dependent sodium channels. Nat Commun 5:3420
Miranda, Pablo; Contreras, Jorge E; Plested, Andrew J R et al. (2013) State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels. Proc Natl Acad Sci U S A 110:5217-22
Lopez-Rodriguez, Angelica; Holmgren, Miguel (2012) Restoration of proper trafficking to the cell surface for membrane proteins harboring cysteine mutations. PLoS One 7:e47693
Gadsby, David C; Bezanilla, Francisco; Rakowski, Robert F et al. (2012) The dynamic relationships between the three events that release individual Na? ions from the Na?/K?-ATPase. Nat Commun 3:669
Galarza-Muñoz, Gaddiel; Soto-Morales, Sonia I; Holmgren, Miguel et al. (2011) Physiological adaptation of an Antarctic Na+/K+-ATPase to the cold. J Exp Biol 214:2164-74

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