Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the ␤␥ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K ؉ channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP 2 and Mg 2؉ -dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated G␣ and G␤␥ subunits was required for gating of the channel. These results confirm the Kir3

Glutamate Dehydrogenase (GDH) is central to the metabolism of glutamate, a major excitatory transmitter in mammalian central nervous system (CNS). hGDH1 is activated by ADP and L-leucine and powerfully inhibited by GTP. Besides this housekeeping hGDH1, duplication led to an hGDH2 isoform that is expressed in the human brain dissociating its function from GTP control. The novel enzyme has reduced basal activity (4-6% of capacity) while remaining remarkably responsive to ADP/L-leucine activation. While the molecular basis of this evolutionary adaptation remains unclear, substitution of Ser for Arg443 in hGDH1 is shown to diminish basal activity (< 2% of capacity) and abrogate L-leucine activation. To explore whether the Arg443Ser mutation disrupts hydrogen bonding between Arg443 and Ser409 of adjacent monomers in the regulatory domain ('antenna'), we replaced Ser409 by Arg or Asp in hGDH1. The Ser409Arg-1 change essentially replicated the Arg443Ser-1 mutation effects. Molecular dynamics simulation predicted that Ser409 and Arg443 of neighboring monomers come in close proximity in the open conformation and that introduction of Ser443-1 or Arg409-1 causes them to separate with the swap mutation (Arg409/Ser443) reinstating their proximity. A swapped Ser409Arg/Arg443Ser-1 mutant protein, obtained in recombinant form, regained most of the wild-type hGDH1 properties. Also, when Ser443 was replaced by Arg443 in hGDH2 (as occurs in hGDH1), the Ser443Arg-2 mutant acquired most of the hGDH1 properties. Hence, side-chain interactions between 409 and 443 positions in the 'antenna' region of hGDHs are crucial for basal catalytic activity, allosteric regulation, and relative resistance to thermal inactivation. Keywords: allosteric regulation, circular dichroism, glutamate dehydrogenase, molecular dynamics simulation, molecular modeling, site-directed mutagenesis. Glutamate dehydrogenase (GDH) (EC 1.4.1.3.) catalyzes the oxidative deamination of glutamate to a-ketoglutarate and ammonia using NAD and/or NADP as cofactors, thus providing a major pathway for the inter-conversion of aamino acids and a-ketoacids. The enzyme is allosterically regulated with GTP and ADP/L-leucine serving as the main endogenous negative and positive modulators, respectively. Studies on rat brain have shown that GDH is mainly expressed in astrocytes, where it is thought to be involved in the metabolism of transmitter glutamate (Aoki et al. 1987). Consistent with this possibility are observations showing that Received August 6, 2014; revised manuscript received November 16, 2014; accepted December 15, 2014. Address correspondence and reprint requests to Andreas Plaitakis, Professor of Neurology University of Crete, School of Health Sciences Section of Medicine, Heraklion, Crete, Greece. E-mail: andreasplaitakis@gmail.comAbbreviations used: GDH, glutamate dehydrogenase; HC, hill coefficient; hGDH1, human glutamate dehydrogenase encoded by the GLUD1 gene; hGDH2, human glutamate dehydrogenase encoded by the GLUD2 gene; CD, circular dichroism; SVD,...

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The hallmark functional property of K ATP (ATP-sensitive potassium) channels is inhibition by intracellular ATP, which binds to a well-defined binding site on Kir6.x subunits and stabilizes the closed conformation of a gate in the channel pore. Numerous inwardly-rectifying potassium (Kir) channels possess an aromatic residue in the 'helix bundle crossing' region, forming the narrowest pore constriction in crystal structures of Kir channels, indicating an important role in channel gating. We have identified a remarkable phenotype of mutant channels carrying a glutamate at this position (F168E). Despite the structural prediction of four glutamates in close proximity, F168E channels are predominantly closed at physiological pH. However, intracellular alkalinization causes rapid and reversible channel activation. These findings suggest that F168E glutamates are uncharged at physiological pH but become deprotonated with a pKa~9, resulting in opening due to mutual repulsion of multiple nearby glutamate sidechains. The K-channel pore scaffold likely brings these glutamates into close proximity, stabilizing the protonated (uncharged) form of the glutamate sidechain, and resulting in a dramatic pKa shift relative to free glutamate. Only at more alkaline pH do the glutamates deprotonate, with their mutual repulsion driving channel opening. Consistent with a role in ATP-mediated channel closure, alkalinization also affects channel sensitivity to ATP. Taken together, these findings demonstrate an engineered (not intrinsic) mechanism of channel gating by pH, and suggest that ATP-mediated gating of Kir6.2 involves conformational rearrangement of the bundle crossing region.

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