Kinetics of PIP2 metabolism and KCNQ2/3 channel regulation studied with a voltage-sensitive phosphatase in living cells

Abstract

The signaling phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) is synthesized in two steps from phosphatidylinositol by lipid kinases. It then interacts with KCNQ channels and with pleckstrin homology (PH) domains among many other physiological protein targets. We measured and developed a quantitative description of these metabolic and protein interaction steps by perturbing the PIP2 pool with a voltage-sensitive phosphatase (VSP). VSP can remove the 5-phosphate of PIP2 with a time constant of τ <300 ms and fully inhibits KCNQ currents in a similar time. PIP2 was then resynthesized from phosphatidylinositol 4-phosphate (PIP) quickly, τ = 11 s. In contrast, resynthesis of PIP2 after activation of phospholipase C by muscarinic receptors took ∼130 s. These kinetic experiments showed that (1) PIP2 activation of KCNQ channels obeys a cooperative square law, (2) the PIP2 residence time on channels is <10 ms and the exchange time on PH domains is similarly fast, and (3) the step synthesizing PIP2 by PIP 5-kinase is fast and limited primarily by a step(s) that replenishes the pool of plasma membrane PI(4)P. We extend the kinetic model for signaling from M1 muscarinic receptors, presented in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910344), with this new information on PIP2 synthesis and KCNQ interaction.

Lack of correlation between surface expression and currents in epileptogenic AB-calmodulin binding domain Kv7.2 potassium channel mutants

“…To assess the impact of PIP 2 sensitivity, the channel was co-expressed in HEK293T cells with a Danio rerio voltage-dependent phosphatase (Dr-VSP). This phosphatase, when activated by strong depolarizations (≥ 100 mV), reduces plasma membrane content of PIP 2 and inhibits Kv7 channels [39,40]. Since the Kv7.2 channel per se does not inactivate, we measured the depolarization-induced current decay caused by the Dr-VSP-mediated PIP 2 depletion.…”

“…The rate of M-current decay is related to the affinity for PIP 2 , such as the faster the rate, the lower the affinity. We applied a simple protocol, described for the first time by Falkenburger and coworkers [40] starting with a depolarized step to −20 mV, a voltage in which the phosphatase is not activated, reveling the basal current level, then, a second pulse to + 100 mV with increasing duration, was applied activating Dr-VSP, and inhibiting the channel (Figure 6(b ) , inset). We compared currents at −20 mV before and after varying lengths of Dr-VSP activation to track the onset of the Dr-VSP effect.…”

“…HEK293T cells were used to perform the experiments with Dr-VSP as described [39,40]. Briefly, Dr-VSP was activated by a 200 ms, jump to + 100 mV, and then the voltage was returned to the holding potential of −20 mV.…”

Combined Phosphoinositide and Ca2+ Signals Mediating Receptor Specificity toward Neuronal Ca2+ Channels

“…72 for a review) to rapidly shuttle phosphoinositides between subcellular compartments. As recently discussed (73), it could then be the phosphoinositide transfer protein transport rate that is stimulated by the "mode 2" receptors rather than the PI 4-kinase rate itself. The recent work using voltage-sensitive PIP 2 phosphatases makes clear that PI(4)P replenishment at the PM is rate-limiting for PIP 2 synthesis (73-75).…”

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

“…This regulation has been explained on the basis of two contrasting mechanisms. (i) There is direct phosphoinositide interaction with specific amino acids in the transport protein, explained either on the basis of charge (8 -13) or presence of canonical lipid binding domains such as pleckstrin homology domains (1,8,14). Common to these direct interaction studies has been the demonstration that mutagenesis of specific amino acids leads to changes in molecular interactions with phosphoinositides that lead to subsequent changes in physiologic channel or transporter activity.…”