Voltage-sensing phosphatase reveals temporal regulation of TRPC3/C6/C7 channels by membrane phosphoinositides

Abstract

TRPC3/C6/C7 channels, a subgroup of classical/canonical TRP channels, are activated by diacylglycerol produced via activation of phospholipase C (PLC)-coupled receptors. Recognition of the physiological importance of these channels has been steadily growing, but the mechanism by which they are regulated remains largely unknown. We recently used a membrane-resident danio rerio voltage-sensing phosphatase (DrVSP) to study TRPC3/C6/C7 regulation and found that the channel activity was controlled by PtdIns(4,5)P(2)-DAG signaling in a self-limiting manner (Imai Y et al., the Journal of Physiology, 2012). In this addendum, we present the advantages of using DrVSP as a molecular tool to study PtdIns(4,5)P(2) regulation. DrVSP should be readily applicable for studying phosphoinositide metabolism-linked channel regulation as well as lipid dynamics. Furthermore, in comparison to other modes of self-limiting ion channel regulation, the regulation of TRPC3/C6/C7 channels seems highly susceptible to activation signal strength, which could potentially affect both open duration and the time to peak activation and inactivation. Dysfunction of such self-limiting regulation may contribute to the pathology of the cardiovascular system, gastrointestinal tract and brain, as these channels are broadly distributed and affected by numerous neurohormonal agonists.

Figure 1. DrVSPs on TRPC currents. (A) top: Exemplar of the voltage-dependence of VMI (r) of TRPC3/C6/C7 currents observed in HEK cells. TRPC6 currents were evoked by external application of the DAG lipase inhibitor RHC80267 (100 μM). r indicates the residual current after depolarization. The red arrow shows the transient inhibition elicited by the depolarization. Bottom: VMI of TRPC6 currents plotted against depolarization pulse amplitude applied in the presence of the indicated DrVSP mutants (n = more than 4). Note that the Q-V curves of the mutants are also shifted leftward [V_1/2(OFF] values for R153Q, T156R and I165R are 16, 73 and 60 mV, respectively). (B) top: Atypical inhibition trace obtained from HEK cells co-transfected with TRPC6 and wild-type DrVSP. Brief depolarizations (+100 mV, 500 ms) were applied every 10 sec (protocol displayed in top), and currents were evoked by CCh (100 μM). Middle and bottom: r and τ-recovery are plotted against stimulus number from the upper trace. The blue dashed line in the middle panel suggests DrVSP-available PtdIns(4,5)P₂ (speculative). The typical trace, averaged r, and averaged τ-recovery data were shown in ref. .

Figure 2. Self-limiting regulatory systems in ion channels. Schemes for channel opening (Ch_o). (A): Voltage-gated sodium channels (rNa_V1.2) in vertebrates open (+) and quickly inactivate (–) in response to depolarization (ψ₁ and ψ₂: difference in the potentials coordinate the V-shaped current trace). (B): High voltage-gated Ca channels (Ca_V1.2) open upon membrane depolarization (ψ₁) and inactivate due to negative feedback regulation wherein Ca²⁺ permeating through the channels inactivates the channels (calcium-dependent inactivation). (C): Ligand-gated cys-loop receptors (P2X1) are activated and then desensitized by the concentration-dependent binding of an agonist (a). Copyright (1996) National Academy of Sciences, USA (D): TRPC3/C6/C7 channels are activated by DAG, a product of PtdIns(4,5)P₂, and the resultant reduction in PtdIns(4,5)P₂ independently inhibits channel opening. Strength linkages with G protein-coupled receptors alter the kinetics in TRPC6 currents (tight and loose linkages are represented by the red and black traces, respectively). The respective current traces were obtained from refs. , , and . (E): Currents obtained under self-limiting conditions exhibit faster inactivation than those obtained under unlocked conditions induced by application of excessive PtdIns(4,5)P₂ through the patch-pipette (TRPC7 currents obtained from ref. 11).