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M 1 muscarinic acetylcholine receptors (mAChRs) may represent a viable target for treatment of disorders involving impaired cognitive function. However, a major limitation to testing this hypothesis has been a lack of highly selective ligands for individual mAChR subtypes. We now report the rigorous molecular characterization of a novel compound, benzylquinolone carboxylic acid (BQCA), which acts as a potent, highly selective positive allosteric modulator (PAM) of the rat M 1 receptor. This compound does not directly activate the receptor, but acts at an allosteric site to increase functional responses to orthosteric agonists. Radioligand binding studies revealed that BQCA increases M 1 receptor affinity for acetylcholine. We found that activation of the M 1 receptor by BQCA induces a robust inward current and increases spontaneous EPSCs in medial prefrontal cortex (mPFC) pyramidal cells, effects which are absent in acute slices from M 1 receptor knock-out mice. Furthermore, to determine the effect of BQCA on intact and functioning brain circuits, multiple single-unit recordings were obtained from the mPFC of rats that showed BQCA increases firing of mPFC pyramidal cells in vivo. BQCA also restored discrimination reversal learning in a transgenic mouse model of Alzheimer's disease and was found to regulate non-amyloidogenic APP processing in vitro, suggesting that M 1 receptor PAMs have the potential to provide both symptomatic and disease modifying effects in Alzheimer's disease patients. Together, these studies provide compelling evidence that M 1 receptor activation induces a dramatic excitation of PFC neurons and suggest that selectively activating the M 1 mAChR subtype may ameliorate impairments in cognitive function.

We have isolated a novel gene, hKCa4, encoding an intermediate conductance, calcium-activated potassium channel from a human lymph node library. The translated protein comprises 427 amino acids, has six transmembrane segments, S1-S6, and a pore motif between S5 and S6. hKCa4 shares 41-42% similarity at the amino acid level with three small conductance calcium-activated potassium channels cloned from brain. Northern blot analysis of primary human T lymphocytes reveals a 2.2-kilobase transcript that is highly up-regulated in activated compared with resting cells, concomitant with an increase in KCa current. hKCa4 transcript is also detected by Northern blots or by polymerase chain reaction in placenta, prostate, thymus, spleen, colon, and many cell lines of hematopoietic origin. Patch-clamp recordings of hKCa4-transfected HEK 293 cells reveal a large voltage-independent, inwardly rectifying potassium current that is blocked by externally applied tetraethylammonium (K d ‫؍‬ 30 ؎ 7 mM), charybdotoxin (K d ‫؍‬ 10 ؎ 1 nM), and clotrimazole (K d ‫؍‬ 387 ؎ 34 nM), but is resistant to apamin, iberiotoxin, kaliotoxin, scyllatoxin (K d > 1 M), and margatoxin (K d > 100 nM). Single hKCa4 channels have a conductance of 33 ؎ 2 picosiemens in symmetrical potassium solutions. The channel is activated by intracellular calcium (K d ‫؍‬ 270 ؎ 8 nM) with a highly cooperative interaction of approximately three calcium ions per channel. These properties of the cloned channel are very similar to those reported for the native KCa channel in activated human T lymphocytes, indicating that hKCa4 encodes this channel type.Potassium channels play a critical role in modulating calcium signaling of lymphocytes (1). Human T lymphocytes express at least two types of potassium channels (2) (8), and B lymphocytes (9), as well as in other peripheral tissues. However, the molecular identity of this channel type was hitherto unknown. We report the cloning and characterization of an intermediate conductance, CTX-sensitive KCa channel, which we call hKCa4, from a human lymph node cDNA library. We present convergent molecular, biophysical, and pharmacological evidence that hKCa4 encodes the predominant KCa channel in human T cells. EXPERIMENTAL PROCEDURESData Base Search-We performed a BLAST search of a proprietary EST data base (licensed from Incyte Pharmaceuticals, Palo Alto, CA) for unannotated potassium channel sequences using the pore sequence of hKv2.1, a Shab-related K ϩ channel (PASFWWATITMTTVGYGDIYP; Ref. 10). Two overlapping clones of interest were identified, and their sequences were determined (Applied Biosystems PRISM™377 automated sequencer). Both of these clones were from a cDNA library of adherent mononuclear cells, which came from a pool of male and female donors.Library Screening and Computer Analysis-A 32 P-labeled DNA fragment from one of the above clones, corresponding to nucleotides 262-1265 in Fig. 1A, was used as a probe to screen ϳ600,000 recombinant plaques from a human lymph node gt10 cDNA library (CLONTECH). Hybridizations w...

The significance of the mismatch negativity (MMN), an event-related potential measured in humans which indexes novelty in the auditory environment, has motivated a search for a cellular correlate of this process. A leading candidate is stimulus-specific adaptation (SSA) in auditory cortex units, which shares several characteristics with the MMN. Whether auditory cortex responses encode sensory novelty, a defining property of the MMN, however, has not been resolved. To evaluate this key issue, we used several variations of the auditory oddball paradigm from the human literature and examined psychophysical and pharmacological properties of multiunit activity in the auditory cortex of awake rodents. We found converging evidence dissociating SSA from sensory novelty and the MMN. First, during an oddball paradigm with frequency deviants, neuronal responses showed clear SSA but failed to encode novelty in a manner analogous to the human MMN. Second, oddball paradigms using intensity or duration deviants revealed a pattern of unit responses that showed sensory adaptation, but again without any measurable novelty correlates aligning to the human MMN. Finally NMDA antagonists, which are known to disrupt the MMN, suppressed the magnitude of multiunit responses in a nonspecific manner, leaving the process of SSA intact. Together, our results suggest that auditory novelty detection as indexed by the MMN is dissociable from SSA at the level of activity encoded by auditory cortex neurons. Further, the NMDA sensitivity reported for the MMN, which models the disruption of MMN observed in schizophrenia, may occur at a mechanistic locus outside of SSA.

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