Background and purpose: Positive modulators of small conductance Ca 2 þ -activated K þ channels (SK1, SK2, and SK3) exert hyperpolarizing effects that influence the activity of excitable and non-excitable cells. The prototype compound 1-EBIO or the more potent compound NS309, do not distinguish between the SK subtypes and they also activate the related intermediate conductance Ca 2 þ -activated K þ channel (IK). This paper demonstrates, for the first time, subtype-selective positive modulation of SK channels. Experimental approach: Using patch clamp and fluorescence techniques we studied the effect of the compound cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) on recombinant hSK1-3 and hIK channels expressed in HEK293 cells. CyPPA was also tested on SK3 and IK channels endogenously expressed in TE671 and HeLa cells. Key results: CyPPA was found to be a positive modulator of hSK3 (EC 50 ¼ 5.6 7 1.6 mM, efficacy 90 7 1.8 %) and hSK2 (EC 50 ¼ 14 7 4 mM, efficacy 71 7 1.8 %) when measured in inside-out patch clamp experiments. CyPPA was inactive on both hSK1 and hIK channels. At hSK3 channels, CyPPA induced a concentration-dependent increase in the apparent Ca 2 þ -sensitivity of channel activation, changing the EC 50 (Ca 2 þ ) from 429 nM to 59 nM. Conclusions and implications: As a pharmacological tool, CyPPA may be used in parallel with the IK/SK openers 1-EBIO and NS309 to distinguish SK3/SK2-from SK1/IK-mediated pharmacological responses. This is important for the SK2 and SK1 subtypes, since they have overlapping expression patterns in the neocortical and hippocampal regions, and for SK3 and IK channels, since they co-express in certain peripheral tissues.

SK channels are small conductance Ca 2ϩ -activated K ϩ channels important for the control of neuronal excitability, the fine tuning of firing patterns, and the regulation of synaptic mechanisms. The classic SK channel pharmacology has largely focused on the peptide apamin, which acts extracellularly by a pore-blocking mechanism.

The ␥-aminobutyric acid, type A (GABA A ) receptor is a chloride-conducting receptor composed of ␣, ␤, and ␥ subunits assembled in a pentameric structure forming a central pore. Each subunit has a large extracellular agonist binding domain and four transmembrane domains (M1-M4), with the second transmembrane (M2) domain lining the pore. Mutation of five amino acids in the M1-M2 loop of the ␤ 3 subunit to the corresponding amino acids of the ␣ 7 nicotinic acetylcholine subunit rendered the GABA A receptor cation-selective upon coexpression with wild type ␣ 2 and ␥ 2 subunits. Similar mutations in the ␣ 2 or ␥ 2 subunits did not lead to such a change in ion selectivity. This suggests a unique role for the ␤ 3 subunit in determining the ion selectivity of the GABA A receptor. The pharmacology of the mutated GABA A receptor is similar to that of the wild type receptor, with respect to muscimol binding, Zn 2؉ and bicuculline sensitivity, flumazenil binding, and potentiation of GABA-evoked currents by diazepam. There was, however, an increase in GABA sensitivity (EC 50 ‫؍‬ 1.3 M) compared with the wild type receptor (EC 50 ‫؍‬ 6.4 M) and a loss of desensitization to GABA of the mutant receptor.The ␥-aminobutyric acid receptor (GABA A R) 1 is a member of the superfamily of ligand-gated ion channels (LGICs), which also includes the nicotinic acetylcholine receptors (nAChR), the glycine receptors (GlyR), and a subtype of the 5-hydroxytryptamine receptors (5-HT 3 R) (1-3). The LGICs are involved in mediating fast neurotransmission in the central nervous system but play different roles, which is reflected in their different ion selectivities. GABA A Rs and GlyRs are anion-selective whereas the nAChRs and 5-HT 3 Rs are cation-selective (3-6). All LGICs are integral membrane proteins, which are formed by assembly of five homologous subunits around a central ion channel (7-10). Each subunit has a large extracellular N-terminal domain and a C-terminal domain containing four transmembrane segments, designated M1-M4, connected by relatively short loops. The extracellular N-terminal domains are believed to form the agonist binding sites, whereas the transmembrane domains form the channel; with the five M2 domains being the primary lining of the ion-conducting pore of the receptor (11,12).The structure of the ion-conducting pore of the LGICs have been described thoroughly using the substituted-cysteine-accessibility method (12-15). All the data support a hypothesis of a predominant ␣-helical structure of the M2 domains with a slight kink in the center. The pore was found to be widest at the extracellular end narrowing toward the cytoplasmic side (12-16). The selectivity filter and gate was found to be at the intracellular end of the M2 domains and to include a part of the M1-M2 loop (16,17). Differences between the individualLGICs were observed, but in general both anions and cations can enter the extracellular vestibules, charge selection occurs at a more intracellular position than 13Ј, and the gate is constituted by amino acids...

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