Cyclic nucleotide-gated (CNG) channels play essential roles in the transduction of visual and olfactory information. They sense variations in the intracellular concentration of cyclic nucleotides that occur in response to visual or olfactory stimuli. CNG channels coassemble as tetramers of homologous subunits, 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. Improper targeting of membrane proteins causes many diseases. Often point mutations to cysteine hinder the delivery of membrane proteins to the cell surface, or to the correct side of polarized cells 8,9. Because cysteine is a readily reactive amino acid, in principle it should be possible to recover proper trafficking by modifying its chemical structure in order to mimic the side chain of the wild type amino acid. As a proof of principle, we have studied two naturally occurring cysteine mutations in a CNG channel (CNGA3) responsible for hereditary cone photoreceptor disorders: Y181C linked to incomplete achromatopsia and R277C linked to complete and incomplete achromatopsia or cone dystrophy. We have chosen these mutations because proper surface CNG channel expression can be easily assayed using electrophysiological techniques, and because both mutations, which cause channel retention in the endoplasmic reticulum (ER), change wild type amino acids of drastically different chemistries. CNG channels open a cationic selective permeation pathway in response to intracellular cyclic nucleotides. Functional homotetramers can be formed by the CNGA1, A2 or A3 subunits, and these channels are usually studied as homotetramers in heterologous systems. We have introduced both achromatopsia-related cysteines in a cysteine-less CNGA1 channel, and used them as a target for specific chemical modification with hydroxybenzyl- (MTSHB) and aminoethyl-methanethiosulfonate (MTSEA). These reagents readily attach to the side chain of cysteines and mimic the chemistry of tyrosine and arginine, respectively. Although Y181C and R277C caused ER retention, after chemical modification we successfully restored both trafficking and normal function to CNGA1 mutant channels R272C and Y176C. Maturation of any protein is a complex, multi-step process involving a network of intracellular proteins and organelles. Surely, all genetic mutations leading to defective maturation cannot be repaired by a single strategy. Thus far, a variety of experimental approaches have been shown to recover proper maturation. For example, cell surface expression of mutant HERG and CNGA3 channels, as well as deficient lysosomal glucocerebrosidase, can be restored simply by lowering the temperature of incubation. Based on their ability to stabilize proper folding conformations in the ER, drugs and lipid chaperones are emerging as new strategies to restore protein maturation. As with other rescue methods, ours has disadvantages: it is restricted to cysteine mutants and it is not specific since MTS reagents will modify any accessible cysteine in a protein. Nevertheless, outside of therapeutics, this method has applications that could, in principle, offer relevant structural and functional information about diseases. For example, it could be used to better understand the chemical nature of the protein folding failure since many different MTS reagents are available that attach moieties resembling different amino acid side chains. Another potential use could be for kinetic studies of folding. For example, if a cysteine mutation impairs proper folding, modification reactions by MTS reagents are sufficiently fast to permit the temporal resolution of downstream conformational changes, providing kinetic information on folding steps. A third application we envision is to use the disease related mutant in the same way that biophysicists use engineered cysteines as a tool to study state dependent accessibility. This will provide information of conformational changes at the site of the cysteine mutation. 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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