Cyclic nucleotide-gated (CNG) channels play essential roles in the transduction of visual and olfactory information (Stryer, 1986;Zufall et al., 1994). They sense variations in the intracellular concentration of cyclic nucleotides that occur in response to visual or olfactory stimuli. In many ways, CNG channels are similar to voltage-activated potassium (Kv) channels. They coassemble as tetramers of homologous subunits (Weitz et al., 2002;Zheng et al., 2002;Zheng and Zagotta, 2004;Zhong et al., 2002), each containing six transmembrane segments (TM), a positively charged TM4 and a reentry P-region between TM5 and TM6, suggesting that CNG channels belong to the same superfamily of voltage-activated cation channels (Jan and Jan, 1990). The main difference is that CNG channels are not gated by changes in membrane voltage. Instead, they open and close the pore in response to changes in the intracellular concentrations of cGMP or cAMP, a property conferred by the presence of a cyclic nucleotide binding domain at the C-terminus of each subunit (Kaupp and Seifert, 2002;Zagotta and Siegelbaum, 1996). Several studies indicate that the pore region of CNG channels play a role in gating. For example, accessibility of cysteine reagents applied from the intracellular (Becchetti et al., 1999;Becchetti and Roncaglia, 2000) and the extracellular (Becchetti et al., 1999;Becchetti and Roncaglia, 2000;Liu and Siegelbaum, 2000) side of the channel to cysteines substituted along the entire P-region have shown that modification of some residues in this region perturb normal gating by cGMP. In a recent study (Contreras et al., 2008), we showed that the gate that opens the permeation pathway in CNG channels is located at the selectivity filter of CNG channels. We came to this conclusion by careful studies assessing the state dependent accessibility to the permeation pathway of intracellular blockers (Contreras and Holmgren, 2006) and also by examining the ability of small thiol specific reagents to chemically modify cysteines substituted along the permeation pathway (Contreras et al., 2008). As we were carrying out this work, we became aware of a unique opportunity to investigate, experimentally, the voltage profile along the permeation pathway of an open channel. In ion channels, the transmembrane potential plays a critical role in ion conduction by acting as a driving force for permeant ions. At the molecular level, the transmembrane potential is thought to decay non linearly across the ion permeation pathway because of its irregular three-dimensional shape. In this study, we determined the voltage profile along an open cyclic nucleotide-gated (CNG) channel by substituting cysteine at three positions known to be in the lining of the permeation pathway (Shi et al., 2006;Contreras et al., 2008): the intracellular cavity, and the inwardly and the outwardly facing ends of the selectivity filter. We evaluated the voltage dependence of modification by small positively charged thiol reagents such as Ag+, Cd2+ and MTSET. Similar to permeant ions, these probes will sense the transmembrane voltage as they transverse the permeation pathway, and therefore changes in the kinetics of the reactions between these thiol reagents and the substituted cysteines can be used to infer the voltage profile along the pore. This approach is feasible as long as the channels gating is not sensitive to voltage, as is the case for CNG channels in the presence of saturating concentrations of cGMP (Benndorf et al., 1999a). By comparing the voltage dependence of the apparent modification rates along the permeation pathway of CNG channels we determined that, as hypothesized, the voltage drops exclusively along the selectivity filter in an open channel. These experimental observations are consistent with electrostatic PB-V calculations performed on a homology model of an open CNG channel based on the high resolution structure of the NaK channel (Alam &Jiang, 2009). 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 (Skou, 1957), the Na/K-ATPase was proposed to alternately transport Na and K ions according to a model known as the Post-Albers scheme (Post et al., 1965;Albers, 1967). As ions are transported through the Na/K pump, they become temporarily occluded within the protein, inaccessible from either side, before being released (Post et al., 1972;Beauge and Glynn, 1979;Glynn et al., 1984;Glynn and Karlish, 1990). By restricting Na/K pumps to only the reversible transitions associated with deocclusion and extracellular release of Na+, Nakao and Gadsby (Nakao and Gadsby, 1986) were able 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 (Rakowski, 1993;Hilgemann, 1994;Holmgren et al., 2000;Peluffo, 2004;Holmgren and Rakowski, 2006). At a fixed membrane potential and external sodium concentration (Nao), 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. This year, we published our first approach to understand the thermodynamic of the Na and K transport by he pump. In this case we took a biological approach, we asked a simple question: how could an Antarctic Na/K pump operate in the extreme cold? Interestingly, we found that the amino acid sequence of polar Na/K ATPases is equipped to operate faster than other pumps at cold temperatures.

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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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