Biophysical physiology of phosphoinositide rapid dynamics and regulation in living cells

Abstract

Phosphoinositide membrane lipids are ubiquitous low-abundance signaling molecules. They direct many physiological processes that involve ion channels, membrane identification, fusion of membrane vesicles, and vesicular endocytosis. Pools of these lipids are continually broken down and refilled in living cells, and the rates of some of these reactions are strongly accelerated by physiological stimuli. Recent biophysical experiments described here measure and model the kinetics and regulation of these lipid signals in intact cells. Rapid on-line monitoring of phosphoinositide metabolism is made possible by optical tools and electrophysiology. The experiments reviewed here reveal that as for other cellular second messengers, the dynamic turnover and lifetimes of membrane phosphoinositides are measured in seconds, controlling and timing rapid physiological responses, and the signaling is under strong metabolic regulation. The underlying mechanisms of this metabolic regulation remain questions for the future.

Figure 1.

Structure of mammalian phosphoinositides. The three yellow positions (R3, R4, and R5) on the myo-inositol ring can be phosphorylated by lipid kinase enzymes and dephosphorylated by lipid phosphatases to yield seven phosphorylated combinations and permutations.

Figure 2.

Synthesis and breakdown of PtdIns(4,5)P₂ (red). This reaction diagram is often called the PI cycle. The lipid species on the gray membrane bar, PtdIns, PtdIns(4)P, etc., are found in the cellular membranes named below in blue. Enzymes are green. Almost all the enzymes have multiple isoforms differing in expression and localization. DAG, diacylglycerol; PM, plasma membrane; R–G_q–PLCβ, receptor-activated PLCβ.

Figure 3.

Transient suppression of M current by a 20-s application of Oxo-M to a neuron from the rat superior cervical sympathetic ganglion. The number labels indicate the time after the start of Oxo-M. The half-recovery time is 89 s (from Suh and Hille, 2002).

Figure 4.

Depletion of PtdIns(4,5)P₂ in response to a muscarinic agonist (Oxo-M) in cell lines. The diamonds show loss of FRET when FRET pairs PH_PLCδ1-CFP and PH_PLCδ1-YFP translocate from being bound near each other at the plasma membrane to being free in the cytoplasm, and the triangles show appearance of PH_PLCδ1-YFP in the cytoplasm from confocal images (transfected tsA201 cells). Circles are PtdInsP₂ from mass spectrometry (mass spec, in CHO-M cells stably expressing M₁ receptors), and colored lines are amplitudes of KCNQ2/3 current (tsA201 cells; Jensen et al., 2009; from Traynor-Kaplan et al., 2017).

Figure 5.

Measuring several steps in the receptor-induced activation of PLCβ and depletion of PtdIns(4,5)P₂ in response to a muscarinic agonist. (A) Cartoon of the signaling pathway from muscarinic agonist to KCNQ2/3 channel. PIP₂, PtdIns(4,5)P₂. (B) YFP-CFP FRET signals, normalized to start at zero, monitor conformational changes within the receptor (intraR, intramolecular FRET between CFP and YFP labels in the receptor), receptor interaction with Gβ, Gα_q interaction with PLCβ, and depletion of PtdIns(4,5)P₂. Note logarithmic time axis. Each line comes from tsA201 cells transfected with M₁ receptors, the appropriate pair of FRET probes, or, in the case of I_KCNQ2/3, with KCNQ2 and KCNQ3 channel subunits. Data assembled from Jensen et al. (2009).

Figure 6.

Testing whether PLCβ is rate-limiting in the receptor-induced hydrolysis of PtdIns(4,5)P₂. KCNQ2/3 current suppression is three times faster in tsA201 cells overexpressing PLCβ. Half-decay times are indicated (from Jensen et al., 2009).

Figure 7.

Summary of half times for PtdIns(4,5)P₂ synthesis and breakdown. The diagram represents some important steps in phosphoinositide metabolism—the PI cycle—and the measured half times for segments we could study in living tsA201 cells at room temperature as explained in the text. Everything might be three to four times faster at 37°C. DAG, diacylglycerol; PI, PtdIns; PI(4)P, PtdIns(4)P; PI(4,5)P₂, PtdIns(4,5)P₂.

Figure 8.

Recovery of PtdIns(4,5)P₂ after depletion. KCNQ2/3 current suppression and recovery are recorded to reflect depletion and recovery of PtdIns(4,5)P₂. Recovery half times are indicated. (A) Diagram of PtdIns(4,5)P₂ resynthesis showing also phosphatases and the voltage-sensitive VSP enzyme. (B) Depletion and recovery after a short application (25 s) of Oxo-M agonist (10 μM). The experiment is similar to that in Fig. 3 except that rather than using a neuron with endogenous receptors and channels, this one uses tsA201 cells transfected with M₁ receptors and KCNQ2 and KCNQ3 channel subunits. PIP₂, PtdIns(4,5)P₂. From Horowitz et al. (2005). (C) A tsA201 cell transfected with channels and VSP (no receptors) is depolarized in a three-step protocol starting at 0 s. The first 1-s step to −20 mV activates KCNQ2/3 current, the next 1-s step to 100 mV activates VSP briefly, and the third step to −20 mV monitors current recovery during the resynthesis of PtdIns(4,5)P₂. The olive trace shows a cell that had also been transfected with PIP 5-kinase. From Falkenburger et al. (2010a).

Figure 9.

Generation of Ins(1,4,5)P₃ and diacylglycerol during prolonged agonist application—and the simultaneous regeneration of PtdIns(4,5)P₂. (A) Representative normalized FRET traces from three cells using C1A domain (diacylglycerol), LIBRAvIII (Ins[1,4,5]P₃), and PH_PLCδ1 (PtdIns[4,5]P₂) probes. (B) Time courses of effective rate constants of lipid kinases from the mathematical model showing upregulation during agonist action. Data and model modified from Myeong et al. (2020).