The bacterial potassium channel KcsA is gated by high concentrations of intracellular protons, allowing the channel to open at pH < 5.5. Despite prior attempts to determine the mechanism responsible for pH gating, the proton sensor has remained elusive. We have constructed a KcsA channel mutant that remains open up to pH 9.0 by replacing key ionizable residues from the N and C termini of KcsA with residues mimicking their protonated counterparts with respect to charge. A series of individual and combined mutations were investigated by using single-channel recordings in lipid bilayers. We propose that these residues are the protonbinding sites and at neutral pH they form a complex network of inter-and intrasubunit salt bridges and hydrogen bonds near the bundle crossing that greatly stabilize the closed state. In our model, these residues change their ionization state at acidic pH, thereby disrupting this network, modifying the electrostatic landscape near the channel gate, and favoring channel opening.ion channel ͉ proton sensor ͉ salt bridge network ͉ pH gating A ctivity of ion channel proteins is modulated by signaling molecules that tightly control the opening and closing of the channel pores, allowing ions to cross the membrane in response to cellular signals. Protons are ubiquitous modulators of ion channel gating and permeation, likely because of the presence of titratable residues located near channel gates, pores, allosteric sites, and regulatory interfaces. Ion channels sensitive to either cytoplasmic or extracellular pH include: transient receptor potential (TRP) and acid-sensing (ASIC) channels, inward rectifier potassium channels (Kir), CLC chloride channels, NMDA receptors, and Ca-activated potassium channels (1-7). Strict modulation of channel gating near neutral pH is often crucial for the physiological roles of these channels. Despite the importance of pH modulation in these channels, the molecular mechanisms of proton gating are not completely understood, partly because of the absence of detailed structural information.The prokaryotic potassium channel KcsA, the first K ϩ channel characterized with x-ray crystallography (8), is modulated by pH in a very narrow acidic pH range (9, 10). The availability of an atomic structure combined with a sensitive functional assay (electrophysiological current recordings with purified channel protein) make KcsA an ideal system for locating and dissecting its pH sensor. KcsA senses pH at its intracellular side (10) and the pH sensor location has been further narrowed by truncation constructs that maintain the pH sensitivity of the full-length channel (11). Moreover, an NMR study recently implicated a histidine located near the bundle crossing of KcsA as the pH sensor (12). Despite major advances in our understanding of this archetypal ion channel, there is no detailed molecular picture of the pH sensor. Elucidating the mechanism underlying KcsA pH sensing may provide a foundation for understanding similar pH-gating dependencies in eukaryotic potassium channels....

Olfactory bulb (OB) glomeruli, the initial sites of synaptic processing of odor information, exhibit high levels of nicotinic acetylcholine receptor (nAChR) expression and receive strong cholinergic input from the basal forebrain. The role of glomerular nAChRs in olfactory processing, however, remains to be elucidated. External tufted (ET) cells are a major source of excitation in the glomerulus and an important component of OB physiology. We have examined the role of nAChRs in modulating ET cell activity using whole-cell electrophysiology in mouse OB slices. We show here that the activation of glomerular nAChRs leads to direct ET cell excitation, as well as an increase in the frequency of spontaneous postsynaptic GABAergic currents. β2-containing nAChRs, likely the α4β2*-nAChR subtype (* represents the possible presence of other subunits), were significant contributors to these effects. The nAChR-mediated increase in spontaneous postsynaptic GABAergic current frequency on ET cells was, for the most part, dependent on glutamate receptor activation, thus implicating a role for excitation-dependent inhibition within the glomerulus. β2-containing nAChRs also regulate the frequency of miniature inhibitory postsynaptic currents on ET cells, implying nicotinic modulation of dendrodendritic signaling between ET and periglomerular cells. Our data also indicate that nAChR activation does not affect spontaneous or evoked transmission at the olfactory nerve-to-ET cell synapse. The results from this study suggest that ET cells, along with mitral cells, play an important role in the nicotinic modulation of glomerular inhibition.

In the mouse olfactory bulb glomerulus, the GABAergic periglomerular (PG) cells provide a major inhibitory drive within the microcircuit. Here we examine GABAergic synapses between these interneurons. At these synapses, GABA is depolarizing and exerts a bimodal control on excitability. In quiescent cells, activation of GABA A receptors can induce the cells to fire, thereby providing a means for amplification of GABA release in the glomerular microcircuit via GABA-induced GABA release. In contrast, GABA is inhibitory in neurons that are induced to fire tonically. PG-PG interactions are modulated by nicotinic acetylcholine receptors (nAChRs), and our data suggest that changes in intracellular calcium concentrations triggered by nAChR activation can be amplified by GABA release. Our results suggest that bidirectional control of inhibition in PG neurons can allow for modulatory inputs, like the cholinergic inputs from the basal forebrain, to determine threshold set points for filtering out weak olfactory inputs in the glomerular layer of the olfactory bulb via the activation of nAChRs.

Nicotinic acetylcholine receptors (nAChRs) regulate information transfer across the main olfactory bulb by instituting a high-pass intensity filter allowing for the filtering out of weak inputs. Excitation-driven inhibition of the glomerular microcircuit via GABA release from periglomerular cells appears to underlie this effect of nAChR activation. The multiplicity of nAChR subtypes and cellular locations raises questions about their respective roles in mediating their effects on the glomerular output. In this study, we address this issue by targeting heteromeric nAChRs using receptor knockouts (KOs) for the two dominant nAChR β-subunit genes known to be expressed in the central nervous system. KOs of the β-nAChR subunit did not affect nAChR currents from mitral cells (MCs) but attenuated those from the external tufted (ET) cells. In slices from these animals, activation of nAChRs still effectively inhibited excitatory postsynaptic currents (EPSCs) and firing on MCs evoked by the olfactory nerve (ON) stimulation, thereby indicating that the filter mechanism was intact. On the other hand, recordings from β-KOs showed that nAChR responses from MCs were abolished and those from ET cells were attenuated. Excitation-driven feedback was abolished as was the effect of nAChR activation on ON-evoked EPSCs. Experiments using calcium imaging showed that one possible consequence of the β-subunit activation might be to alter the time course of calcium transients in juxtaglomerular neurons suggesting a role for these receptors in calcium signaling. Our results indicate that nAChRs containing the β-subunit are critical in the filtering of odor inputs and play a determinant role in the cholinergic modulation of glomerular output. NEW & NOTEWORTHY In this study, using receptor gene knockouts we examine the relative contributions of heteromeric nAChR subtypes located on different cell types to this effect of receptor activation. Our results demonstrate that nAChRs containing the β-subunit activate MCs resulting in feedback inhibition from glomerular interneurons. This period of inhibition results in the selective filtering of weak odor inputs providing one mechanism by which nAChRs can enhance discrimination between two closely related odors.