7TM pharmacology measured by label-free: a holistic approach to cell signalling.

Ligands acting at 7-transmembrane receptors (7TMs) transduce effects on cellular behaviour in a notion termed efficacy; in turn, the cellular behaviour or phenotype can be quantified. Underpinning efficacy is the ability of ligands to dictate the triggering of distinct intracellular signalling event(s) in a system-dependent manner through selective stabilisation of receptor conformations. Given the wealth of putative cell signalling routes a receptor species may possess (spectrum of activities) and numerous mechanisms by which ligand-receptor pairings signal, the call for an integrated solution to cellular activity has come to light. The potential of novel methodologies to probe for 7TM function such as label-free has been subjected to much attention in recent years. Label-free detection differs greatly from the arsenal of the so-called traditional 7TM techniques commonly employed. It provides a temporally resolved cumulative readout of cellular activity using intact and living cells. It holds vast promise in enabling cellular behaviours to be estimated in a global or 'holistic' manner. This article will focus on key 7TM areas of interest where label-free has been particularly impactful of late rather than covering the principles behind the methodologies (which have been reviewed elsewhere). Firstly, it has facilitated the detection of endogenous or native-like cellular systems that are possibly more physiologically relevant; secondly, it has offered unprecedented angles to the probing of functional selectivity and ligand efficacy.

Pharmacological characterisation of the orexin receptor subtype mediating postsynaptic excitation in the rat dorsal raphe nucleus.

Electrophysiological recordings from dorsal raphe nucleus (DRN) neurones in rat brain slices have revealed that the orexins can cause direct and reversible depolarisation of the postsynaptic membrane. Whilst it is known that the membrane depolarisation produced by orexin-A can dramatically increase the firing rate of DRN neurones, quantitative pharmacological analysis that determines the receptor subtype mediating the orexinergic response has not yet been performed. Here, we demonstrate that the rank order of potencies of orexin receptor agonists to excite serotonergic DRN neurones is orexin-A = orexin-B > SB-668875-DM. In contrast, the rank order of potency of these agonists to excite noradrenergic locus coreleus (LC) neurones is orexin-A > orexin-B > SB-668875-DM. We show further that the orexin receptor antagonist, SB-334867-A, inhibits the effects of orexin-A in the LC and DRN with pKB values of 6.93 and 5.84, respectively, values similar to those calculated for human OX1 (7.27) and OX2 (5.60) receptors expressed in CHO cells. These data suggest a differential role for OX1 and OX2 receptors in stimulating distinct populations of monoaminergic neurones in the rat CNS with OX2 receptors exhibiting a more pronounced functional significance in serotonergic neurones and OX1 in noradrenergic neurones.

Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives.

We have studied activation by phorbol derivatives of TRPV4 channels, the human VRL-2, and murine TRP12 channels, which are highly homologous to the human VR-OAC, and the human and murine OTRPC4 channel. 4alpha-Phorbol 12,13-didecanoate (4alpha-PDD) induced an increase in intracellular Ca(2+) concentration, [Ca(2+)](i), in 1321N1 cells stably transfected with human VRL-2 (hVRL-2.1321N1) or HEK-293 cells transiently transfected with murine TRP12, but not in nontransfected or mock-transfected cells. Concomitantly with the increase in [Ca(2+)](i), 4alpha-PDD activated an outwardly rectifying cation channel with an Eisenman IV permeation sequence for monovalent cations that is Ca(2+)-permeable with P(Ca)/P(Na) = 5.8. Phorbol 12-myristate 13-acetate also induced an increase in [Ca(2+)](i) but was approximately 50 times less effective than 4alpha-PDD. EC(50) for Ca(2+) increase and current activation was nearly identical (pEC(50) approximately 6.7). Similar effects were observed in freshly isolated mouse aorta endothelial cells which express TRP12 endogenously. By using 4alpha-PDD as a tool to stimulate TRP12, we showed that activation of this channel is modulated by [Ca(2+)](i); an increase in [Ca(2+)](i) inhibits the channel with an IC(50) of 406 nm. Ruthenium Red at a concentration of 1 microm completely blocks inward currents at -80 mV but has a smaller effect on outward currents likely indicating a voltage dependent channel block. We concluded that the phorbol derivatives activate TRPV4 (VR-OAC, VRL-2, OTRPC4, TRP12) independently from protein kinase C, in a manner consistent with direct agonist gating of the channel.