David R. Piper

Characterization of SB-269970-A, a selective 5-HT7 receptor antagonist

The novel 5‐HT7 receptor antagonist, SB‐269970‐A, potently displaced [3H]‐5‐CT from human 5‐HT7(a) (pKi 8.9±0.1) and 5‐HT7 receptors in guinea‐pig cortex (pKi 8.3±0.2). 5‐CT stimulated adenylyl cyclase activity in 5‐HT7(a)/HEK293 membranes (pEC50 7.5±0.1) and SB‐269970‐A (0.03–1 μM) inhibited the 5‐CT concentration‐response with no significant alteration in the maximal response. The pA2 (8.5±0.2) for SB‐269970‐A agreed well with the pKi determined from [3H]‐5‐CT binding studies. 5‐CT‐stimulated adenylyl cyclase activity in guinea‐pig hippocampal membranes (pEC50 of 8.4±0.2) was inhibited by SB‐269970‐A (0.3 μM) with a pKB (8.3±0.1) in good agreement with its antagonist potency at the human cloned 5‐HT7(a) receptor and its binding affinity at guinea‐pig cortical membranes. 5‐HT7 receptor mRNA was highly expressed in human hypothalamus, amygdala, thalamus, hippocampus and testis. SB‐269970‐A was CNS penetrant (steady‐state brain : blood ratio of ca. 0.83 : 1 in rats) but was rapidly cleared from the blood (CLb=ca. 140 ml min−1 kg−1). Following a single dose (3 mg kg−1) SB‐269970 was detectable in rat brain at 30 (87 nM) and 60 min (58 nM). In guinea‐pigs, brain levels averaged 31 and 51 nM respectively at 30 and 60 min after dosing, although the compound was undetectable in one of the three animals tested. 5‐CT (0.3 mg kg−1 i.p.) induced hypothermia in guinea‐pigs was blocked by SB‐269970‐A (ED50 2.96 mg kg−1 i.p.) and the non‐selective 5‐HT7 receptor antagonist metergoline (0.3–3 mg kg−1 s.c.), suggesting a role for 5‐HT7 receptor stimulation in 5‐CT induced hypothermia in guinea‐pigs. SB‐269970‐A (30 mg kg−1) administered at the start of the sleep period, significantly reduced time spent in Paradoxical Sleep (PS) during the first 3  h of EEG recording in conscious rats.

Gating currents associated with intramembrane charge displacement in HERG potassium channels

HERG (human ether-a-go-go-related gene) encodes a delayed rectifier K+ channel vital to normal repolarization of cardiac action potentials. Attenuation of repolarizing K+ current caused by mutations in HERG or channel block by common medications prolongs ventricular action potentials and increases the risk of arrhythmia and sudden death. The critical role of HERG in maintenance of normal cardiac electrical activity derives from its unusual gating properties. Opposite to other voltage-gated K+ channels, the rate of HERG channel inactivation is faster than activation and appears to be intrinsically voltage dependent. To investigate voltage sensor movement associated with slow activation and fast inactivation, we characterized HERG gating currents. When the cut-open oocyte voltage clamp technique was used, membrane depolarization elicited gating current with fast and slow components that differed 100-fold in their kinetics. Unlike previously studied voltage-gated K+ channels, the bulk of charge movement in HERG was protracted, consistent with the slow rate of ionic current activation. Despite similar kinetic features, fast inactivation was not derived from the fast gating component. Analysis of an inactivation-deficient mutant HERG channel and a Markov kinetic model suggest that HERG inactivation is coupled to activation.

Identification and characterization of neuronal precursors and their progeny from human fetal tissue

We have examined primary human neuronal precursors (HNPs) from 18–22‐week‐old fetuses. We showed that E‐NCAM/MAP2/β‐III tubulin‐immunoreactive neuronal precursors divide in vitro and could be induced to differentiate into mature neurons in 2 weeks. HNPs did not express nestin and differentiated slowly compared to rodent neuronal restricted precursors (NRPs, 5 days). Immunocytochemical and physiological analyses showed that HNPs could generate a heterogeneous population of neurons that expressed neurofilament‐associated protein and various neurotransmitters, neurotransmitter synthesizing enzymes, voltage‐gated ion channels, and ligand‐gated neurotransmitter receptors and could fire action potentials. Undifferentiated and differentiated HNPs did not coexpress glial markers. Only a subset of cells that expressed GFP under the control of the Tα1 tubulin promoter was E‐NCAM/β‐III tubulin‐immunoreactive, indicating nonexclusive overlap between these two HNP cell populations. Overall, HNPs resemble NRPs isolated from rodent tissue and appear to be a neuronal precursor population. J. Neurosci. Res. 66:356–368, 2001.

Calcium Signalling in Squid Olfactory Receptor Neurons

Isolated squid olfactory receptor neurons respond to dopamine and betaine with hyperpolarizing conductances. We used Ca²⁺ imaging techniques to determine if changes in intracellular Ca²⁺ were involved in transducing the hyperpolarizing odor responses. We found that dopamine activated release of Ca²⁺ from intracellular stores while betaine did not change internal Ca²⁺ concentrations. Application of 10 mM caffeine also released Ca²⁺ from intracellular stores, suggesting the presence of ryanodine-like receptors. Depletion of intracellular stores with 100 μM thapsigargin revealed the presence of a Ca²⁺ store depletion-activated Ca²⁺ influx. The influx of Ca²⁺ through the store-operated channel was reversibly blocked by 10 mM Cd²⁺. Taken together, these data suggest a novel odor transduction system in squid olfactory receptor neurons involving Ca²⁺ release from intracellular stores.

Residues in the first transmembrane domain of the Caenorhabditis elegans GABAA receptor confer sensitivity to the neurosteroid pregnenolone sulfate

1 The GABAA receptor is a target of endogenous and synthetic neurosteroids. Little is known about the residues required for neurosteroid action on GABAA receptors. We have investigated pregnenolone sulfate (PS) inhibition of the Caenorhabditis elegans UNC‐49 GABA receptor, a close homolog of the mammalian GABAA receptor. 2 The UNC‐49 locus encodes two GABA receptor subunits, UNC‐49B and UNC‐49C. UNC‐49C is sensitive to PS but UNC‐49B is not sensitive. By analyzing chimeric receptors and receptors containing site‐directed mutations, we identified two regions required for PS inhibition. 3 Four residues in the first transmembrane domain are required for the majority of the sensitivity to PS, but a charged extracellular residue at the end of the M2 helix also plays a role. Strikingly, mutation of one additional M1 residue reverses the effect of PS from an inhibitor to an enhancer of receptor function. 4 Mutating the M1 domain had little effect on sensitivity to the inhibitor picrotoxin, suggesting that these residues may mediate neurosteroid action specifically, and not allosteric regulation in general.

Cooperative interactions between R531 and acidic residues in the voltage sensing module of hERG1 channels

HERG1 K+ channels are critical for modulating the duration of the cardiac action potential. The role of hERG1 channels in maintaining electrical stability in the heart derives from their unusual gating properties: slow activation and fast inactivation. HERG1 channel inactivation is intrinsically voltage sensitive and is not coupled to activation in the same way as in the Shaker family of K+ channels. We recently proposed that the S4 transmembrane domain functions as the primary voltage sensor for hERG1 activation and inactivation and that distinct regions of S4 contribute to each gating process. In this study, we tested the hypothesis that S4 rearrangements underlying activation and inactivation gating may be associated with distinct cooperative interactions between a key residue in the S4 domain (R531) and acidic residues in neighboring regions (S1 – S3 domains) of the voltage sensing module. Using double-mutant cycle analysis, we found that R531 was energetically coupled to all acidic residues in S1-S3 during activation, but was coupled only to acidic residues near the extracellular portion of S2 and S3 (D456, D460 and D509) during inactivation. We propose that hERG1 activation involves a cooperative conformational change involving the entire voltage sensing module, while inactivation may involve a more limited interaction between R531 and D456, D460 and D509.

Voltage Sensing and Activation Gating of HCN Pacemaker Channels

Activation of pacemaker channels underlie the spontaneous diastolic depolarization of sinoatrial node cells in the heart. Four similar genes encoding these hyperpolarization-activated, cyclic nucleotide-gated channels were recently cloned and subsequently named HCN1-4. Here we review the physiological role of HCN channels and recent findings regarding mechanisms of channel gating. Like all other voltage-gated channels, site-directed mutagenesis analysis indicates that the highly charged S4 transmembrane domain is the voltage sensor. However, unlike most other channels channel, opening occurs in response to membrane hyperpolarization rather than depolarization.