contributed equally to this work Thyroid hormone, acting through several nuclear hormone receptors, plays important roles in thermogenesis, lipogenesis and maturation of the neonatal brain. The receptor specificity for mediating these effects is largely unknown, and to determine this we developed mice lacking the thyroid hormone receptor TRα1. The mice have an average heart rate 20% lower than that of control animals, both under normal conditions and after thyroid hormone stimulation. Electrocardiograms show that the mice also have prolonged QRS-and QT end -durations. The mice have a body temperature 0.5°C lower than normal and exhibit a mild hypothyroidism, whereas their overall behavior and reproduction are normal. The results identify specific and important roles for TRα1 in regulation of tightly controlled physiological functions, such as cardiac pacemaking, ventricular repolarisation and control of body temperature.

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains three domains: the hydrophilic domains I and III harbor the binding sites for NAD(H) and NADP(H), respectively, and domain II represents the membrane-spanning region. Proton translocation involves primarily domain II but possibly also domain III, which contains the essential betaAsp392 residue. In the present investigation, the major portions of domain I (EcTHSalpha1 and EcTHSalpha2) and domain III (EcTHSbeta1) were overexpressed in E. coli and purified therefrom. EcTHSbeta1 was purified mainly in its holoform containing approximately 95% NADP+ and 5% NADPH. When combined, EcTHSalpha1/EcTHSalpha2 and EcTHSbeta1 were catalytically active, indicating native-like structures. Due to the lack of structural information and its possible role in proton pumping, EcTHSbeta1 was primarily characterized. Substrate-binding characteristics and conformational changes were investigated by fluorescence and CD. Fluorescence arising from the single betaTrp415 of EcTHSbeta1 was quenched upon binding of NADPH by resonance energy transfer, an effect that provides an important tool for investigating substrate interactions with this domain and the determination of Kd values. The apparent relative binding affinity for NADPH was found to be about 50 times higher than that for NADP+. Circular dichroism was used to estimate secondary structure content and for conformational analysis of EcTHSbeta1 in the absence and presence of added substrates at various temperatures. Results show that domain III complexed with NADPH or NADP+ adopts different conformations. Isoelectric focusing and native gel electrophoresis experiments support this finding. It is proposed that these structural differences play a central role in a conformationally-driven proton pump mechanism of the intact enzyme.

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We have used a telemetry system to record heart rate, body temperature, electrocardiogram (ECG), and locomotor activity in awake, freely moving mice lacking thyroid hormone receptor (TR)-β or TR-α1 and -β (TR-α1/β). The TR-α1/β-deficient mice had a reduced heart rate compared with wild-type controls. The TR-β-deficient mice showed an elevated heart rate, which, however, was unresponsive to thyroid hormone treatment regardless of hormonal serum levels. ECG revealed that the TR-β-deficient mice had a shortened Q-Tend time in contrast to the TR-α1/β-deficient mice, which exhibited prolonged P-Q and Q-Tend times. Mental or pharmacological stimulation of the sympathetic nervous system resulted in a parallel increase in heart rate in all animals. A single injection of a nonselective β-adrenergic-receptor blocker resulted in a parallel decrease in all mice. The TR-α1/β-deficient mice also had a 0.4°C lower body temperature than controls, whereas no difference was observed in locomotor activity between the different strains of mice. Our present and previous results support the hypothesis that TR-α1 has a major role in determining heart rate under baseline conditions and body temperature and that TR-β mediates a hormone-induced increase in heart rate.