Jorge E. Sánchez-Rodríguez

Ouabain binding site in a functioning Na+/K+ ATPase

Background: Ouabain binds at the permeation pathway of the Na+/K+ ATPase. Results: We have identified two binding sites for ouabain along the ion conductive pathway of the Na+/K+ pump that are mutually exclusive and differ in their affinities by about an order of magnitude. Conclusion: Ouabain reaches its high affinity binding site at the inner end of the permeation pathway by a sequential mechanism. Significance: This work unifies all available functional and structural data on the interactions of ouabain with the Na+/K+ pump. The Na+/K+ ATPase is an almost ubiquitous integral membrane protein within the animal kingdom. It is also the selective target for cardiotonic derivatives, widely prescribed inhibitors for patients with heart failure. Functional studies revealed that ouabain-sensitive residues distributed widely throughout the primary sequence of the protein. Recently, structural work has brought some consensus to the functional observations. Here, we use a spectroscopic approach to estimate distances between a fluorescent ouabain and a lanthanide binding tag (LBT), which was introduced at five different positions in the Na+/K+ ATPase sequence. These five normally functional LBT-Na+/K+ ATPase constructs were expressed in the cell membrane of Xenopus laevis oocytes, operating under physiological internal and external ion conditions. The spectroscopic data suggest two mutually exclusive distances between the LBT and the fluorescent ouabain. From the estimated distances and using homology models of the LBT-Na+/K+ ATPase constructs, approximate ouabain positions could be determined. Our results suggest that ouabain binds at two sites along the ion permeation pathway of the Na+/K+ ATPase. The external site (low apparent affinity) occupies the same region as previous structural findings. The high apparent affinity site is, however, slightly deeper toward the intracellular end of the protein. Interestingly, in both cases the lactone ring faces outward. We propose a sequential ouabain binding mechanism that is consistent with all functional and structural studies.

β1-subunit–induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel

Significance Large-conductance Ca2+- and voltage-activated K+ (BK) channels play many physiological roles, ranging from the maintenance of smooth muscle tone to the modulation of alcohol tolerance. In most cases, this physiological versatility of the BK channel is due to the association of the pore-forming α-subunit with β-subunits. Therefore, it is of importance to know what the structural consequences of this association are. Here, using lanthanide-based resonance energy transfer, we were able to determine the extracellular position of transmembrane segments S0–S2 with and without the β1-subunit and the position of the two transmembrane segments of the β1 subunit in the α/β1-subunit complex. We concluded that β1 produces rearrangements of the BK voltage sensor domain. Large-conductance Ca2+- and voltage-activated K+ (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit–induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.

A Structural Rearrangement of the Na+/K+-ATPase Traps Ouabain within the External Ion Permeation Pathway

With the use of the energy of ATP hydrolysis, the Na+/K+-ATPase is able to transport across the cell membrane Na+ and K+ against their electrochemical gradients. The enzyme is strongly inhibited by ouabain and its derivatives, some that are therapeutically used for patients with heart failure (cardiotonic steroids). Using lanthanide resonance energy transfer, we trace here the conformational changes occurring on the external side of functional Na+/K+-ATPases induced by the binding of ouabain. Changes in donor/acceptor pair distances are mainly observed within the α subunit of the enzyme. To derive a structural model matching the experimental lanthanide resonance energy transfer distances measured with bound ouabain, we carried out molecular dynamics simulations with energy restraints applied simultaneously using a novel methodology with multiple non-interacting fragments. The restrained simulation, initiated from the X-ray structure of the E2(2K+) state, became strikingly similar to the X-ray structure of the sodium-bound state. The final model shows that ouabain is trapped within the external ion permeation pathway of the pump.

Development of a PET radioligand for potassium channels to image CNS demyelination

Central nervous system (CNS) demyelination represents the pathological hallmark of multiple sclerosis (MS) and contributes to other neurological conditions. Quantitative and specific imaging of demyelination would thus provide critical clinical insight. Here, we investigated the possibility of targeting axonal potassium channels to image demyelination by positron emission tomography (PET). These channels, which normally reside beneath the myelin sheath, become exposed upon demyelination and are the target of the MS drug, 4-aminopyridine (4-AP). We demonstrate using autoradiography that 4-AP has higher binding in non-myelinated and demyelinated versus well-myelinated CNS regions, and describe a fluorine-containing derivative, 3-F-4-AP, that has similar pharmacological properties and can be labeled with 18F for PET imaging. Additionally, we demonstrate that [18F]3-F-4-AP can be used to detect demyelination in rodents by PET. Further evaluation in Rhesus macaques shows higher binding in non-myelinated versus myelinated areas and excellent properties for brain imaging. Together, these data indicate that [18F]3-F-4-AP may be a valuable PET tracer for detecting CNS demyelination noninvasively.

LRET Determination of Molecular Distances during pH Gating of the Mammalian Inward Rectifier Kir1.1b

Gating of the mammalian inward rectifier Kir1.1 at the helix bundle crossing (HBC) by intracellular pH is believed to be mediated by conformational changes in the C-terminal domain (CTD). However, the exact motion of the CTD during Kir gating remains controversial. Crystal structures and single-molecule fluorescence resonance energy transfer of KirBac channels have implied a rigid body rotation and/or a contraction of the CTD as possible triggers for opening of the HBC gate. In our study, we used lanthanide-based resonance energy transfer on single-Cys dimeric constructs of the mammalian renal inward rectifier, Kir1.1b, incorporated into anionic liposomes plus PIP2, to determine unambiguous, state-dependent distances between paired Cys residues on diagonally opposite subunits. Functionality and pH dependence of our proteoliposome channels were verified in separate electrophysiological experiments. The lanthanide-based resonance energy transfer distances measured in closed (pH 6) and open (pH 8) conditions indicated neither expansion nor contraction of the CTD during gating, whereas the HBC gate widened by 8.8 ± 4 Å, from 6.3 ± 2 to 15.1 ± 6 Å, during opening. These results are consistent with a Kir gating model in which rigid body rotation of the large CTD around the permeation axis is correlated with opening of the HBC hydrophobic gate, allowing permeation of a 7 Å hydrated K ion.