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

Falkenburger BH, Jensen JB, Hille B - J. Gen. Physiol. (2010)

Bottom Line: These kinetic experiments showed that (1) PIP(2) activation of KCNQ channels obeys a cooperative square law, (2) the PIP(2) residence time on channels is <10 ms and the exchange time on PH domains is similarly fast, and (3) the step synthesizing PIP(2) 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 M(1) muscarinic receptors, presented in our companion paper in this issue (Falkenburger et al. 2010.Physiol. doi:10.1085/jgp.200910344), with this new information on PIP(2) synthesis and KCNQ interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
The signaling phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP(2)) 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 PIP(2) pool with a voltage-sensitive phosphatase (VSP). VSP can remove the 5-phosphate of PIP(2) with a time constant of tau <300 ms and fully inhibits KCNQ currents in a similar time. PIP(2) was then resynthesized from phosphatidylinositol 4-phosphate (PIP) quickly, tau = 11 s. In contrast, resynthesis of PIP(2) after activation of phospholipase C by muscarinic receptors took approximately 130 s. These kinetic experiments showed that (1) PIP(2) activation of KCNQ channels obeys a cooperative square law, (2) the PIP(2) residence time on channels is <10 ms and the exchange time on PH domains is similarly fast, and (3) the step synthesizing PIP(2) 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 M(1) 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 PIP(2) synthesis and KCNQ interaction.

Show MeSH

Related in: MedlinePlus

KCNQ2/3 current behaves like the square of PH probe FRET. (A) In four cells, single exponentials were fitted to simultaneously acquired KCNQ2/3 current and PH probe FRETr during VSP activation (2 s of +100 mV). Measurements from the same cell are connected by a line. (B) In five cells, single exponentials were fitted to simultaneously acquired KCNQ2/3 current and PH probe FRETr during recovery after VSP activation. KCNQ2/3 current was measured as tail current amplitude. (C) VSP effect on KCNQ current with voltage protocol as in Fig. 3 B. Recovery of KCNQ2/3 current at −20 mV was fitted with a double exponential: y = a − b*exp(−c*t) + d*exp(−f*t). (Inset) Summary of time constants from 31 cells. Time constant of the positive term (pos.) is 1/f, and that of the negative term (neg.) is 1/c. (D) Illustration of the consequence of squaring an exponential of the form y = 1 − exp(−t/τ). (E) Plot of KCNQ2/3 current at −20 mV (black) versus PH probe FRETr at the same time during recovery after VSP activation in the cell depicted in Fig. 2. Similar observations were made for three other cells. Red curve corresponds to the equation given. (F) Averaged KCNQ2/3 current at −20 mV versus averaged FRETr at the same time after M1R activation, measured in separate cells (data from Figs. 5 D and 7 B in Jensen et al., 2009). (G) Illustration of the dependence of FRETr (approximated by PH_PIP2; see Fig. S3) and KCNQ current on PIP2 concentration as predicted by the model outlined in Fig. 7 and Tables I and II: Kd of PH probe is 2,000 µm−2 for PIP2 and 0.1 µM for IP3 (0.16 µM IP3); Kd of KCNQ is 2,000 µm−2 for PIP2. KCNQ current = (KCNQ_PIP2)2.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2812502&req=5

fig4: KCNQ2/3 current behaves like the square of PH probe FRET. (A) In four cells, single exponentials were fitted to simultaneously acquired KCNQ2/3 current and PH probe FRETr during VSP activation (2 s of +100 mV). Measurements from the same cell are connected by a line. (B) In five cells, single exponentials were fitted to simultaneously acquired KCNQ2/3 current and PH probe FRETr during recovery after VSP activation. KCNQ2/3 current was measured as tail current amplitude. (C) VSP effect on KCNQ current with voltage protocol as in Fig. 3 B. Recovery of KCNQ2/3 current at −20 mV was fitted with a double exponential: y = a − b*exp(−c*t) + d*exp(−f*t). (Inset) Summary of time constants from 31 cells. Time constant of the positive term (pos.) is 1/f, and that of the negative term (neg.) is 1/c. (D) Illustration of the consequence of squaring an exponential of the form y = 1 − exp(−t/τ). (E) Plot of KCNQ2/3 current at −20 mV (black) versus PH probe FRETr at the same time during recovery after VSP activation in the cell depicted in Fig. 2. Similar observations were made for three other cells. Red curve corresponds to the equation given. (F) Averaged KCNQ2/3 current at −20 mV versus averaged FRETr at the same time after M1R activation, measured in separate cells (data from Figs. 5 D and 7 B in Jensen et al., 2009). (G) Illustration of the dependence of FRETr (approximated by PH_PIP2; see Fig. S3) and KCNQ current on PIP2 concentration as predicted by the model outlined in Fig. 7 and Tables I and II: Kd of PH probe is 2,000 µm−2 for PIP2 and 0.1 µM for IP3 (0.16 µM IP3); Kd of KCNQ is 2,000 µm−2 for PIP2. KCNQ current = (KCNQ_PIP2)2.

Mentions: To quantitate the kinetics of VSP actions on KCNQ2/3 current, we switched to measuring current by holding continuously at −20 mV, where noninactivating KCNQ2/3 current can be maintained for a long time and other endogenous K+ currents in tsA201 cells are minimally activated (Fig. S2, A and D). PH probes were not expressed in these experiments. VSP was activated by brief steps to +100 mV, a perturbation that also increased the driving force for K+ and, in cells without VSP, increased current through KCNQ2/3 and endogenous channels (Fig. 3 A). When Dr-VSP was coexpressed (Fig. 3 B), KCNQ2/3 current decayed during the +100-mV depolarization. Current was much reduced upon return to −20 mV and recovered thereafter. We compared currents at −20 mV before and after varying lengths of VSP activation to track the onset of the VSP effect (Fig. 3, C and D). The effect on KCNQ2/3 was maximal after a 1-s activation pulse. A half-maximal effect required ∼120 ms, and significant effect was already evident with only 40 ms of VSP activation, just enough time for 63% movement of the voltage sensor charge (Fig. S1 A). Thus, the coupling of the VSP voltage sensor to the phosphatase activity and the reporting of PIP2 depletion by KCNQ2/3 current are both fast (<40 ms). Fitting exponentials to the decline of whole cell current at +100 mV (Fig. 4 A) gave a time course for the action of VSP consistent with that of Fig. 3 D, even though this measure would be contaminated by the slow activation of KCNQ2/3 current and the inactivation of endogenous currents at +100 mV (Fig. S2, A and B).


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

Falkenburger BH, Jensen JB, Hille B - J. Gen. Physiol. (2010)

KCNQ2/3 current behaves like the square of PH probe FRET. (A) In four cells, single exponentials were fitted to simultaneously acquired KCNQ2/3 current and PH probe FRETr during VSP activation (2 s of +100 mV). Measurements from the same cell are connected by a line. (B) In five cells, single exponentials were fitted to simultaneously acquired KCNQ2/3 current and PH probe FRETr during recovery after VSP activation. KCNQ2/3 current was measured as tail current amplitude. (C) VSP effect on KCNQ current with voltage protocol as in Fig. 3 B. Recovery of KCNQ2/3 current at −20 mV was fitted with a double exponential: y = a − b*exp(−c*t) + d*exp(−f*t). (Inset) Summary of time constants from 31 cells. Time constant of the positive term (pos.) is 1/f, and that of the negative term (neg.) is 1/c. (D) Illustration of the consequence of squaring an exponential of the form y = 1 − exp(−t/τ). (E) Plot of KCNQ2/3 current at −20 mV (black) versus PH probe FRETr at the same time during recovery after VSP activation in the cell depicted in Fig. 2. Similar observations were made for three other cells. Red curve corresponds to the equation given. (F) Averaged KCNQ2/3 current at −20 mV versus averaged FRETr at the same time after M1R activation, measured in separate cells (data from Figs. 5 D and 7 B in Jensen et al., 2009). (G) Illustration of the dependence of FRETr (approximated by PH_PIP2; see Fig. S3) and KCNQ current on PIP2 concentration as predicted by the model outlined in Fig. 7 and Tables I and II: Kd of PH probe is 2,000 µm−2 for PIP2 and 0.1 µM for IP3 (0.16 µM IP3); Kd of KCNQ is 2,000 µm−2 for PIP2. KCNQ current = (KCNQ_PIP2)2.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812502&req=5

fig4: KCNQ2/3 current behaves like the square of PH probe FRET. (A) In four cells, single exponentials were fitted to simultaneously acquired KCNQ2/3 current and PH probe FRETr during VSP activation (2 s of +100 mV). Measurements from the same cell are connected by a line. (B) In five cells, single exponentials were fitted to simultaneously acquired KCNQ2/3 current and PH probe FRETr during recovery after VSP activation. KCNQ2/3 current was measured as tail current amplitude. (C) VSP effect on KCNQ current with voltage protocol as in Fig. 3 B. Recovery of KCNQ2/3 current at −20 mV was fitted with a double exponential: y = a − b*exp(−c*t) + d*exp(−f*t). (Inset) Summary of time constants from 31 cells. Time constant of the positive term (pos.) is 1/f, and that of the negative term (neg.) is 1/c. (D) Illustration of the consequence of squaring an exponential of the form y = 1 − exp(−t/τ). (E) Plot of KCNQ2/3 current at −20 mV (black) versus PH probe FRETr at the same time during recovery after VSP activation in the cell depicted in Fig. 2. Similar observations were made for three other cells. Red curve corresponds to the equation given. (F) Averaged KCNQ2/3 current at −20 mV versus averaged FRETr at the same time after M1R activation, measured in separate cells (data from Figs. 5 D and 7 B in Jensen et al., 2009). (G) Illustration of the dependence of FRETr (approximated by PH_PIP2; see Fig. S3) and KCNQ current on PIP2 concentration as predicted by the model outlined in Fig. 7 and Tables I and II: Kd of PH probe is 2,000 µm−2 for PIP2 and 0.1 µM for IP3 (0.16 µM IP3); Kd of KCNQ is 2,000 µm−2 for PIP2. KCNQ current = (KCNQ_PIP2)2.
Mentions: To quantitate the kinetics of VSP actions on KCNQ2/3 current, we switched to measuring current by holding continuously at −20 mV, where noninactivating KCNQ2/3 current can be maintained for a long time and other endogenous K+ currents in tsA201 cells are minimally activated (Fig. S2, A and D). PH probes were not expressed in these experiments. VSP was activated by brief steps to +100 mV, a perturbation that also increased the driving force for K+ and, in cells without VSP, increased current through KCNQ2/3 and endogenous channels (Fig. 3 A). When Dr-VSP was coexpressed (Fig. 3 B), KCNQ2/3 current decayed during the +100-mV depolarization. Current was much reduced upon return to −20 mV and recovered thereafter. We compared currents at −20 mV before and after varying lengths of VSP activation to track the onset of the VSP effect (Fig. 3, C and D). The effect on KCNQ2/3 was maximal after a 1-s activation pulse. A half-maximal effect required ∼120 ms, and significant effect was already evident with only 40 ms of VSP activation, just enough time for 63% movement of the voltage sensor charge (Fig. S1 A). Thus, the coupling of the VSP voltage sensor to the phosphatase activity and the reporting of PIP2 depletion by KCNQ2/3 current are both fast (<40 ms). Fitting exponentials to the decline of whole cell current at +100 mV (Fig. 4 A) gave a time course for the action of VSP consistent with that of Fig. 3 D, even though this measure would be contaminated by the slow activation of KCNQ2/3 current and the inactivation of endogenous currents at +100 mV (Fig. S2, A and B).

Bottom Line: These kinetic experiments showed that (1) PIP(2) activation of KCNQ channels obeys a cooperative square law, (2) the PIP(2) residence time on channels is <10 ms and the exchange time on PH domains is similarly fast, and (3) the step synthesizing PIP(2) 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 M(1) muscarinic receptors, presented in our companion paper in this issue (Falkenburger et al. 2010.Physiol. doi:10.1085/jgp.200910344), with this new information on PIP(2) synthesis and KCNQ interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
The signaling phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP(2)) 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 PIP(2) pool with a voltage-sensitive phosphatase (VSP). VSP can remove the 5-phosphate of PIP(2) with a time constant of tau <300 ms and fully inhibits KCNQ currents in a similar time. PIP(2) was then resynthesized from phosphatidylinositol 4-phosphate (PIP) quickly, tau = 11 s. In contrast, resynthesis of PIP(2) after activation of phospholipase C by muscarinic receptors took approximately 130 s. These kinetic experiments showed that (1) PIP(2) activation of KCNQ channels obeys a cooperative square law, (2) the PIP(2) residence time on channels is <10 ms and the exchange time on PH domains is similarly fast, and (3) the step synthesizing PIP(2) 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 M(1) 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 PIP(2) synthesis and KCNQ interaction.

Show MeSH
Related in: MedlinePlus