Prestin, a member of the solute carrier family 26, is expressed in the basolateral membrane of outer hair cells. This protein provides the molecular basis for outer hair cell somatic electromotility, which is crucial for the frequency selectivity and sensitivity of mammalian hearing. It has long been known that there are abundantly expressed ϳ11-nM protein particles present in the basolateral membrane. These particles were hypothesized to be the motor proteins that drive electromotility. Because the calculated size of a prestin monomer is too small to form an ϳ11-nM particle, the possibility of prestin oligomerization was examined. We investigated possible quaternary structures of prestin by lithium dodecyl sulfate-PAGE, perfluoro-octanoate-PAGE, a membrane-based yeast two-hybrid system, and chemical cross-linking experiments. Prestin, obtained from different host or native cells, is resistant to dissociation by lithium dodecyl sulfate and behaves as a stable oligomer on lithium dodecyl sulfate-PAGE. In the membrane-based yeast two-hybrid system, homo-oligomeric interactions between prestin-bait/prestinprey suggest that prestin molecules can associate with each other. Chemical cross-linking experiments, perfluoro-octanoate-PAGE/Western blot, and affinity purification experiments all indicate that prestin exists as a higher order oligomer, such as a tetramer, in prestin-expressing yeast, mammalian cell lines and native outer hair cells. Our data from experiments using hydrophobic and hydrophilic reducing reagents suggest that the prestin dimer is connected by a disulfide bond embedded in the prestin hydrophobic core. This stable dimer may act as the building block for producing the higher order oligomers that form the ϳ11-nM particles in the outer hair cell's basolateral membrane.Hearing impairment, the most common congenital sensory defect, affects millions of people from newborns to senior citizens, resulting in large hearing-related health care costs (1). Causes of hearing impairment are often associated with damage to outer hair cells (OHCs). 2 These sensory receptor cells, located in the mammalian organ of Corti, rapidly change their length (2) and stiffness (3) at acoustic frequencies when their transmembrane voltage is altered. Corresponding to this somatic cell-length change, OHCs exhibit voltage-dependent non-linear capacitance (4). This unique somatic electromotility is thought to provide active mechanical amplification of the cochlear response to sound (5). It has long been known that large (ϳ11-nM diameter) membrane protein particles constitute a substantial portion of the lateral membrane in OHCs (6). It is suspected that these abundantly expressed particles are the "motor proteins" responsible for somatic electromotility (7).Prestin, the OHC motor protein (8), is located at the same location where the 11-nM protein particles are found, i.e. in the lateral membrane of OHCs (9 -11). When prestin is heterologously expressed in several mammalian cell lines, the prestinexpressing cells demonstrate all of...

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SignificanceCystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a chloride channel located in the apical membrane of epithelia cells. The cAMP signaling pathway and protein phosphorylation are known to be primary controlling mechanisms for channel function. In this study, we present an alternative activation pathway that involves calcium-activated calmodulin binding of the intrinsically disordered regulatory (R) region of CFTR. Beyond their potential therapeutic value, these data provide insights into the intersection of calcium signaling with control of ion homeostasis and the ways in which the local CFTR microdomain organizes itself.

Glucose is an essential source of energy for the brain. Recently, the development of genetically encoded fluorescent biosensors has allowed real time visualization of glucose dynamics from individual neurons and astrocytes. A major difficulty for this approach, even for ratiometric sensors, is the lack of a practical method to convert such measurements into actual concentrations in ex vivo brain tissue or in vivo. Fluorescence lifetime imaging provides a strategy to overcome this. In a previous study, we reported the lifetime glucose sensor iGlucoSnFR‐TS (then called SweetieTS) for monitoring changes in neuronal glucose levels in response to stimulation. This genetically encoded sensor was generated by combining the Thermus thermophilus glucose‐binding protein with a circularly permuted variant of the monomeric fluorescent protein T‐Sapphire. Here, we provide more details on iGlucoSnFR‐TS design and characterization, as well as pH and temperature sensitivities. For accurate estimation of glucose concentrations, the sensor must be calibrated at the same temperature as the experiments. We find that when the extracellular glucose concentration is in the range 2–10 mM, the intracellular glucose concentration in hippocampal neurons from acute brain slices is ~20% of the nominal external glucose concentration (~0.4–2 mM). We also measured the cytosolic neuronal glucose concentration in vivo, finding a range of ~0.7–2.5 mM in cortical neurons from awake mice.

The CbiT and CbiE enzymes participate in the biosynthesis of vitamin B12. They are fused together in some organisms to form a protein called CobL, which catalyzes two methylations and one decarboxylation on a precorrin intermediate. Because CbiE has sequence homology to canonical precorrin methyltransferases, CbiT was hypothesized to catalyze the decarboxylation. We herein present the crystal structure of MT0146, the CbiT homolog from Methanobacterium thermoautotrophicum. The protein shows structural similarity to Rossmann-like S-adenosyl-methionine-dependent methyltransferases, and our 1.9 A cocrystal structure shows that it binds S-adenosyl-methionine in standard geometry near a binding pocket that could accommodate a precorrin substrate. Therefore, MT0146/CbiT probably functions as a precorrin methyltransferase and represents the first enzyme identified with this activity that does not have the canonical precorrin methyltransferase fold.

The solute carrier gene family 26 (SLC26) encodes membrane proteins with diverse physiological roles but with the common feature of halide involvement. Here, we present bioinformatic and biochemical evidence that SLC26 proteins have intrinsically disordered regions (IDRs) in their C-terminal domains and that these regions contain calmodulin (CaM) binding sites. The veracity of these predictions and the functional consequences of CaM binding were examined in prestin, SLC26A5, as a model for the SLC26 family and as one of the most investigated and best understood members. We found that CaM binds directly to the IDR in the C-terminal domain of prestin in a calcium-obligate manner. Using both isolated murine outer hair cells (OHCs) and a heterologous expression system, we also found that this calcium-obligate CaM binding shifts the operating point of the protein to more hyperpolarized potentials with consequent alteration of the function of the prestin. Because calcium is the main intracellular second messenger used by the efferent medial olivocochlear (MOC) pathway of the auditory system and CaM is abundant in OHCs, the CaM-prestin interaction may be involved in the MOC-mediated modulation of cochlear amplification. However, this regulatory mechanism is not likely to be restricted to cochlear OHCs, in light of both clear bioinformatic evidence and the fact that calcium and CaM are ubiquitous intracellular second messengers used by virtually all cell types. Hence, the calcium/CaM-dependent regulatory mechanism described herein is likely applicable to most, if not all, SLC26 paralogs.

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Highlights d An engineered sensor can detect glucose in living animals with a good signal-to-noise ratio d The sensor can be used to screen molecules that modulate glucose transporter function d The sensor reveals a rostral-caudal glucose transport pathway in fly larval CNS d Two-color imaging shows effects of drugs on glucose transport in living zebrafish