Protein kinase A-independent modulation of ion channels in the brain by cyclic AMP.

Ion channels underlying the electrical activity of neurons can be regulated by neurotransmitters via two basic mechanisms: ligand binding and covalent modification. Whereas neurotransmitters often act by binding directly to ion channels, the intracellular messenger cyclic AMP is thought usually to act indirectly, by activating protein kinase A, which in turn can phosphorylate channel proteins. Here we show that cyclic AMP, and transmitters acting via cyclic AMP, can act in a protein kinase A-independent manner in the brain. In hippocampal pyramidal cells, cyclic AMP and norepinephrine were found to cause a depolarization by enhancing the hyperpolarization-activated mixed cation current, IQ (also called Ih). This effect persisted even after protein kinase A activity was blocked, thus strongly suggesting a kinase-independent action of cyclic AMP. The modulation of this current by ascending monoaminergic fibers from the brainstem is likely to be a widespread mechanism, participating in the state control of the brain during arousal and attention.

A signature of the T → R transition in human hemoglobin

Allosteric effects in hemoglobin arise from the equilibrium between at least two energetic states of the molecule: a tense state, T, and a relaxed state, R. The two states differ from each other in the number and energy of the interactions between hemoglobin subunits. In the T state, constraints between subunits oppose the structural changes resulting from ligand binding. In the R state, these constraints are released, thus enhancing ligand-binding affinity. In the present work, we report the presence of four sites in hemoglobin that are structurally stabilized in the R relative to the T state. These sites are Hisα103(G10) and Hisα122(H5) in each α subunit of hemoglobin. They are located at the α1β1 and α2β2 interfaces of the hemoglobin tetramer, where the histidine side chains form hydrogen bonds with specific residues from the β chains. We have measured the solvent exchange rates of side chain protons of Hisα103(G10) and Hisα122(H5) in both deoxygenated and ligated hemoglobin by NMR spectroscopy. The exchange rates were found to be higher in the deoxygenated-T than in ligated-R state. Analysis of exchange rates in terms of the local unfolding model revealed that the structural stabilization free energy at each of these two histidines is larger by ≈1.5 kcal/(mol tetramer) in the R relative to the T state. The location of these histidines at the intradimeric α1β1 and α2β2 interfaces also suggests a role for these interfaces in the allosteric equilibrium of hemoglobin.