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.

It has been shown that the voltage (V m ) dependence of ClC Cl

(4AP) is a specific blocker of voltage-gated potassium channels (K V 1 family) clinically approved for the symptomatic treatment of patients with multiple sclerosis (MS). It has recently been shown that [ 18 F]3F4AP, a radiofluorinated analog of 4AP, also binds to K V 1 channels and can be used as a PET tracer for the detection of demyelinated lesions in rodent models of MS. Here, we investigate four novel 4AP derivatives containing methyl (-CH 3), methoxy (-OCH 3) as well as trifluoromethyl (-CF 3) in the 2 and 3 position as potential candidates for PET imaging and/or therapy. We characterized the physicochemical properties of these compounds (basicity and lipophilicity) and analyzed their ability to block Shaker K + channel under different voltage and pH conditions. Our results demonstrate that three of the four derivatives are able to block voltage-gated potassium channels. Specifically, 3-methyl-4-aminopyridine (3Me4AP) was found to be approximately 7-fold more potent than 4AP and 3F4AP; 3-methoxy-and 3-trifluoromethyl-4-aminopyridine (3MeO4AP and 3CF 3 4AP) were found to be about 3-to 4-fold less potent than 4AP; and 2-trifluoromethyl-4-AP (2CF 3 4AP) was found to be about 60-fold less active. these results suggest that these novel derivatives are potential candidates for therapy and imaging.

Key points• Plasma membrane ClC-2 chloride channels are widely distributed in our body and are important for vision and fertility.• ClC-2 channels are gated by changes in transmembrane voltage despite of lacking a voltage sensor device. It has been hypothesized that the interaction of an external proton with the gating machinery is responsible for voltage-dependent gating.

Background: Ouabain binds at the permeation pathway of the Na ϩ /K ϩ ATPase.

No abstract

Large-conductance Ca 2+ -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.lanthanide resonance energy transfer | BK channels | β1-subunit I mportant physiological processes involve Ca 2+ entry into cells mediated by voltage-dependent Ca 2+ channels. This divalent cation influx is essential for life because it permits, for example, the adequate functioning of smooth muscle or neurosecretion to occur. Some mechanism must be put into action, however, to control Ca 2+ influx, either to dampen or to stop the physiological effects of the cytoplasmic increase in Ca 2+. In many cases, this dampening mechanism is accomplished by one of the most broadly expressed channels in mammals: the large-conductance Ca 2+ -and voltage-activated K + (BK) channel (1-3). Because there is a single gene coding for the BK channel (Slowpoke KNCMA1), channel diversity must be a consequence of alternative splicing and/or interaction with regulatory subunits. In fact, both mechanisms account for BK channel diversity, but the most dramatic changes in BK channel properties are brought about through the interaction with regulatory subunits, membrane-integral proteins, denominated BK β-subunits (β1-β4) (4-7) and the recently discovered γ-subunits (γ1-γ4) (8, 9).Structurally, the BK channel is a homotetramer of its poreforming α-subunit and is a member of the voltage-dependent potassium (Kv) channel family. Distinct from Kv channels, however, BK channel subunits are composed of seven transmembrane domains S0-S6 (10, 11). Little is known about the detailed structure of the membrane-spanning portion of the BK channel, or of the α/β1-subunit complex. Here, we used a variant of Förster resonance energy transfer (FRET), called lanthanide-based resonance energy transfer (LRET), to determine the positions of the N terminus (NT) and S0, S1, and S2 transmembrane segments of the α-subunit of the BK channel, as well as the position of the β1-subunit in the α/β1-subunit complex. LRET uses luminescent lanthanides (e.g., Tb 3+ ) as donor instead of conventional fluorophores. This technique has been successfully used to measure intramolecular distances and to...

The widely expressed two-pore homodimeric inward rectifier CLC-2 chloride channel regulates transepithelial chloride transport, extracellular chloride homeostasis, and neuronal excitability. Each pore is independently gated at hyperpolarized voltages by a conserved pore glutamate. Presumably, exiting chloride ions push glutamate outwardly while external protonation stabilizes it. To understand the mechanism of mouse CLC-2 opening we used homology modelling-guided structure–function analysis. Structural modelling suggests that glutamate E213 interacts with tyrosine Y561 to close a pore. Accordingly, Y561A and E213D mutants are activated at less hyperpolarized voltages, re-opened at depolarized voltages, and fast and common gating components are reduced. The double mutant cycle analysis showed that E213 and Y561 are energetically coupled to alter CLC-2 gating. In agreement, the anomalous mole fraction behaviour of the voltage dependence, measured by the voltage to induce half-open probability, was strongly altered in these mutants. Finally, cytosolic acidification or high extracellular chloride concentration, conditions that have little or no effect on WT CLC-2, induced reopening of Y561 mutants at positive voltages presumably by the inward opening of E213. We concluded that the CLC-2 gate is formed by Y561-E213 and that outward permeant anions open the gate by electrostatic and steric interactions.