Enhanced Fluorine-19 MRI Sensitivity using a Cryogenic Radiofrequency Probe: Technical Developments and Ex Vivo Demonstration in a Mouse Model of Neuroinflammation.

Neuroinflammation can be monitored using fluorine-19 (19F)-containing nanoparticles and 19F MRI. Previously we studied neuroinflammation in experimental autoimmune encephalomyelitis (EAE) using room temperature (RT) 19F radiofrequency (RF) coils and low spatial resolution 19F MRI to overcome constraints in signal-to-noise ratio (SNR). This yielded an approximate localization of inflammatory lesions. Here we used a new 19F transceive cryogenic quadrature RF probe ( 19 F-CRP) that provides the SNR necessary to acquire superior spatially-resolved 19F MRI. First we characterized the signal-transmission profile of the 19 F-CRP. The 19 F-CRP was then benchmarked against a RT 19F/1H RF coil. For SNR comparison we used reference compounds including 19F-nanoparticles and ex vivo brains from EAE mice administered with 19F-nanoparticles. The transmit/receive profile of the 19 F-CRP diminished with increasing distance from the surface. This was counterbalanced by a substantial SNR gain compared to the RT coil. Intraparenchymal inflammation in the ex vivo EAE brains was more sharply defined when using 150 µm isotropic resolution with the 19 F-CRP, and reflected the known distribution of EAE histopathology. At this spatial resolution, most 19F signals were undetectable using the RT coil. The 19 F-CRP is a valuable tool that will allow us to study neuroinflammation with greater detail in future in vivo studies.

Solution structure and function of the "tandem inactivation domain" of the neuronal A-type potassium channel Kv1.4.

Cumulative inactivation of voltage-gated (Kv) K(+) channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kvalpha subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40-50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.

A helical region in the C terminus of small-conductance Ca2+-activated K+ channels controls assembly with apo-calmodulin.

Small conductance Ca(2+)-activated potassium (SK) channels underlie the afterhyperpolarization that follows the action potential in many types of central neurons. SK channels are voltage-independent and gated solely by intracellular Ca(2+) in the submicromolar range. This high affinity for Ca(2+) results from Ca(2+)-independent association of the SK alpha-subunit with calmodulin (CaM), a property unique among the large family of potassium channels. Here we report the solution structure of the calmodulin binding domain (CaMBD, residues 396-487 in rat SK2) of SK channels using NMR spectroscopy. The CaMBD exhibits a helical region between residues 423-437, whereas the rest of the molecule lacks stable overall folding. Disruption of the helical domain abolishes constitutive association of CaMBD with Ca(2+)-free CaM, and results in SK channels that are no longer gated by Ca(2+). The results show that the Ca(2+)-independent CaM-CaMBD interaction, which is crucial for channel function, is at least in part determined by a region different in sequence and structure from other CaM-interacting proteins.