TRP channels and lipids: from Drosophila to mammalian physiology

Abstract

The transient receptor potential (TRP) ion channel family was the last major ion channel family to be discovered. The prototypical member (dTRP) was identified by a forward genetic approach in Drosophila, where it represents the transduction channel in the photoreceptors, activated downstream of a Gq-coupled PLC. In the meantime 29 vertebrate TRP isoforms are recognized, distributed amongst seven subfamilies (TRPC, TRPV, TRPM, TRPML, TRPP, TRPA, TRPN). They subserve a wide range of functions throughout the body, most notably, though by no means exclusively, in sensory transduction and in vascular smooth muscle. However, their precise physiological roles and mechanism of activation and regulation are still only gradually being revealed. Most TRP channels are subject to multiple modes of regulation, but a common theme amongst the TRPC/V/M subfamilies is their regulation by lipid messengers. Genetic evidence supports an excitatory role of diacylglycerol (DAG) for the dTRP's, although curiously only DAG metabolites (PUFAs) have been found to activate the Drosophila channels. TRPC2,3,6 and 7 are widely accepted as DAG-activated channels, although TRPC3 can also be regulated via a store-operated mechanism. More recently PIP2 has been shown to be required for activity of TRPV5, TRPM4,5,7 and 8, whilst it may inhibit TRPV1 and the dTRPs. Although compelling evidence for a direct interaction of DAG with the TRPC channels is lacking, mutagenesis studies have identified putative PIP2-interacting domains in the C-termini of several TRPV and TRPM channels.

Figure 1. Genetic evidence for the excitatory role of DAG

Mutations in DAG kinase (DGK, rdgA gene) greatly facilitate responses in PLC (norpA gene) and Gαq hypomorphs. A, a bright flash in a severe PLC mutant (norpA^P12) only elicits a few sporadic 1–2 pA ‘quantum bumps’, but in the double mutant norpA^P12,rdgA¹, the response to the same intensity is enhanced ∼100-fold. B, summary of averaged data (mean ± s.e.m.) for macroscopic response amplitude in two norpA alleles (P12 and P16) and also the Gαq mutant (left bar, single mutant; right bar, in double mutant combination with rdgA); note the logarithmic scale. Reference to the roles of PLC and DGK shows how the data can be readily interpreted if it is assumed that DAG is an excitatory messenger. For further details, see Hardie et al. (2002).

Figure 2. PIP₂ in Drosophila photoreceptors monitored by PIP₂ biosensors (Kir2.1)

A, dose–response function of Kir2.1^R228Q channels expressed in Drosophila S2 cells to exogenous di-C₈ PIP₂, applied to inside-out patches (sample response in inset). The R228Q point mutation reduces the effective affinity for PIP₂ by a factor of ∼5 making the channels more sensitive to changes in physiological levels of PIP₂. Arrow indicates approximate effective resting (maximum) level of PIP₂ in the photoreceptors, implying that the current should be an approximately linear indicator of PIP₂ levels in vivo. B, currents mediated by Kir2.1^R228Q channels genetically targeted to the microvillar membrane in trpl:trp double mutants lacking both light-sensitive channels. In the dark a large (∼1 nA) constitutive current is activated by the prevailing PIP₂ levels (extent indicated by double arrow). Calibrated light flashes (0.03, etc. expressed in effectively absorbed photons per microvillus) suppress the current due to hydrolysis of PIP₂ by PLC: flashes containing 1 photon per microvillus deplete the entire cell of the majority of detectable PIP₂ within ∼1 s. The Kir2.1 current is reactivated with a half-time of ∼60 s in the dark, representing the resynthesis of PIP₂. C, intensity response function of PLC activity, expressed as percentage total PIP₂-sensitive Kir2.1 current suppressed per second, measured from the maximum slope of individual flash responses as in B. D, basal PLC activity monitored in the dark in vivo as a function of time after establishing the whole-cell configuration: a control (WT) cell recorded in the dark with ATP in the patch pipette shows only very gradual rundown of the Kir2.1 current over 10 min. Without ATP, however, the current decays to near-zero within ∼6 min. A similar behaviour is seen in the rdgA (DG kinase) mutant demonstrating ongoing basal PLC activity in this mutant as well. Adapted from Hardie et al. 2004.