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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.

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Activation of VSP (Dr-VSP) reduces PH probe FRET. (A) Cells were transfected with PIP2-binding PH probes (PH-PLCδ1) fused to CFP or YFP, Dr-VSP, and KCNQ2 and KCNQ3 channel subunits and recorded in whole cell voltage clamp. (B) Principle of PIP2 measurement by PH probe FRET (see Results and Fig. S3). (C) Photometry setup. Excitation light was scanned from 300 to 500 nm in 200 ms, every 500 ms, and reflected by a dichroic mirror around 440 and 500 nm. Emission light was separated into channels for CFP emission (480/40 nm) and YFP emission (535/30 nm). Time courses (D, E, F, and H) were acquired simultaneously. (D) CFP emission with 440-nm excitation (CFPC). (E) YFP emission with 440-nm excitation, corrected for CFP emission at 535/30 nm and for direct excitation of YFP by 440-nm excitation light (YFPC). (F) YFP emission with 500-nm excitation (YFPY). (G) FRETr = YFPC/CFPC. (H) Tail current amplitude. Membrane was held at −60 mV and depolarized to −20 mV for 300 ms every 500 ms, except for shaded area where membrane was held at +100 mV for 2 s. Tail currents were measured during slow channel deactivation at −60 mV. (I) Time constants of single-exponential fits to FRETr while membrane was held at +100 mV (onset of VSP effect). A summary of 14 cells is shown. (J) Time constant of single-exponential fits to recovery of FRETr after 2 s at +100 mV. A summary of 12 cells is shown.
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fig2: Activation of VSP (Dr-VSP) reduces PH probe FRET. (A) Cells were transfected with PIP2-binding PH probes (PH-PLCδ1) fused to CFP or YFP, Dr-VSP, and KCNQ2 and KCNQ3 channel subunits and recorded in whole cell voltage clamp. (B) Principle of PIP2 measurement by PH probe FRET (see Results and Fig. S3). (C) Photometry setup. Excitation light was scanned from 300 to 500 nm in 200 ms, every 500 ms, and reflected by a dichroic mirror around 440 and 500 nm. Emission light was separated into channels for CFP emission (480/40 nm) and YFP emission (535/30 nm). Time courses (D, E, F, and H) were acquired simultaneously. (D) CFP emission with 440-nm excitation (CFPC). (E) YFP emission with 440-nm excitation, corrected for CFP emission at 535/30 nm and for direct excitation of YFP by 440-nm excitation light (YFPC). (F) YFP emission with 500-nm excitation (YFPY). (G) FRETr = YFPC/CFPC. (H) Tail current amplitude. Membrane was held at −60 mV and depolarized to −20 mV for 300 ms every 500 ms, except for shaded area where membrane was held at +100 mV for 2 s. Tail currents were measured during slow channel deactivation at −60 mV. (I) Time constants of single-exponential fits to FRETr while membrane was held at +100 mV (onset of VSP effect). A summary of 14 cells is shown. (J) Time constant of single-exponential fits to recovery of FRETr after 2 s at +100 mV. A summary of 12 cells is shown.

Mentions: Data were analyzed offline in IGOR Pro 6.0 (WaveMetrics). To calculate FRET, we extracted three values from each wavelength scan, similar to a three-cube FRET approach. We denote them CFPC, raw YFPC, and YFPY, with the first part referring to the emission wavelength and the subscript referring to the excitation wavelength. For the CFPC value, emission in the short-wavelength channel (460–480-nm emission) was integrated over the time when excitation was 360–460 nm. For the raw YFPC value, the long-wavelength channel (535/30-nm emission) was integrated over the same time. For the YFPY value, the long-wavelength channel was integrated over the time where excitation was 490–500 nm. The units for all three values were set as arbitrary fluorescence units (AFU). Background was subtracted from each. The raw YFPC value had to be corrected for cyan fluorescent protein (CFP) emission collected in the long-wavelength channel and for direct excitation of yellow fluorescent protein (YFP) by 440-nm light by subtracting 0.834*CFPC and 0.065*YFPY. The corrected value is referred to as YFPC from now on. The correction factors were determined by measuring cells expressing only CFP or YFP. The spectral window for collection of CFP emission was smaller than in our previous work (Jensen et al., 2009). Therefore CFPC had to be multiplied by a larger factor in correcting the long-wavelength channel for bleedthrough of CFP emission. The lower values for CFPC also affected the values of the FRET ratio, FRETr (see below). FRET was expressed as the ratio FRETr = YFPC/CFPC. This ratio is related to FRET efficiency, with two differences. A FRET efficiency of 20% means that 20% of CFP excitation is reemitted by YFP instead of CFP, thus CFP emission is reduced to 80%. If the short-wavelength detector (CFPC) and the long-wavelength detector (YFPC and YFPY) had the same photon sensitivity, a FRET efficiency of 20% would correspond to a FRETr of 20/80 = 0.25. However, in the photometry setup used here, the absolute changes in YFPC were approximately threefold larger than the accompanying changes in CFPC (both in ΔAFU; compare Fig. 2, D with E). A true FRET efficiency of 20% would therefore correspond to a FRETr of 3*20/80 = 0.75. We report FRETr in arbitrary units because the absolute values will differ between setups.


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)

Activation of VSP (Dr-VSP) reduces PH probe FRET. (A) Cells were transfected with PIP2-binding PH probes (PH-PLCδ1) fused to CFP or YFP, Dr-VSP, and KCNQ2 and KCNQ3 channel subunits and recorded in whole cell voltage clamp. (B) Principle of PIP2 measurement by PH probe FRET (see Results and Fig. S3). (C) Photometry setup. Excitation light was scanned from 300 to 500 nm in 200 ms, every 500 ms, and reflected by a dichroic mirror around 440 and 500 nm. Emission light was separated into channels for CFP emission (480/40 nm) and YFP emission (535/30 nm). Time courses (D, E, F, and H) were acquired simultaneously. (D) CFP emission with 440-nm excitation (CFPC). (E) YFP emission with 440-nm excitation, corrected for CFP emission at 535/30 nm and for direct excitation of YFP by 440-nm excitation light (YFPC). (F) YFP emission with 500-nm excitation (YFPY). (G) FRETr = YFPC/CFPC. (H) Tail current amplitude. Membrane was held at −60 mV and depolarized to −20 mV for 300 ms every 500 ms, except for shaded area where membrane was held at +100 mV for 2 s. Tail currents were measured during slow channel deactivation at −60 mV. (I) Time constants of single-exponential fits to FRETr while membrane was held at +100 mV (onset of VSP effect). A summary of 14 cells is shown. (J) Time constant of single-exponential fits to recovery of FRETr after 2 s at +100 mV. A summary of 12 cells is shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
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fig2: Activation of VSP (Dr-VSP) reduces PH probe FRET. (A) Cells were transfected with PIP2-binding PH probes (PH-PLCδ1) fused to CFP or YFP, Dr-VSP, and KCNQ2 and KCNQ3 channel subunits and recorded in whole cell voltage clamp. (B) Principle of PIP2 measurement by PH probe FRET (see Results and Fig. S3). (C) Photometry setup. Excitation light was scanned from 300 to 500 nm in 200 ms, every 500 ms, and reflected by a dichroic mirror around 440 and 500 nm. Emission light was separated into channels for CFP emission (480/40 nm) and YFP emission (535/30 nm). Time courses (D, E, F, and H) were acquired simultaneously. (D) CFP emission with 440-nm excitation (CFPC). (E) YFP emission with 440-nm excitation, corrected for CFP emission at 535/30 nm and for direct excitation of YFP by 440-nm excitation light (YFPC). (F) YFP emission with 500-nm excitation (YFPY). (G) FRETr = YFPC/CFPC. (H) Tail current amplitude. Membrane was held at −60 mV and depolarized to −20 mV for 300 ms every 500 ms, except for shaded area where membrane was held at +100 mV for 2 s. Tail currents were measured during slow channel deactivation at −60 mV. (I) Time constants of single-exponential fits to FRETr while membrane was held at +100 mV (onset of VSP effect). A summary of 14 cells is shown. (J) Time constant of single-exponential fits to recovery of FRETr after 2 s at +100 mV. A summary of 12 cells is shown.
Mentions: Data were analyzed offline in IGOR Pro 6.0 (WaveMetrics). To calculate FRET, we extracted three values from each wavelength scan, similar to a three-cube FRET approach. We denote them CFPC, raw YFPC, and YFPY, with the first part referring to the emission wavelength and the subscript referring to the excitation wavelength. For the CFPC value, emission in the short-wavelength channel (460–480-nm emission) was integrated over the time when excitation was 360–460 nm. For the raw YFPC value, the long-wavelength channel (535/30-nm emission) was integrated over the same time. For the YFPY value, the long-wavelength channel was integrated over the time where excitation was 490–500 nm. The units for all three values were set as arbitrary fluorescence units (AFU). Background was subtracted from each. The raw YFPC value had to be corrected for cyan fluorescent protein (CFP) emission collected in the long-wavelength channel and for direct excitation of yellow fluorescent protein (YFP) by 440-nm light by subtracting 0.834*CFPC and 0.065*YFPY. The corrected value is referred to as YFPC from now on. The correction factors were determined by measuring cells expressing only CFP or YFP. The spectral window for collection of CFP emission was smaller than in our previous work (Jensen et al., 2009). Therefore CFPC had to be multiplied by a larger factor in correcting the long-wavelength channel for bleedthrough of CFP emission. The lower values for CFPC also affected the values of the FRET ratio, FRETr (see below). FRET was expressed as the ratio FRETr = YFPC/CFPC. This ratio is related to FRET efficiency, with two differences. A FRET efficiency of 20% means that 20% of CFP excitation is reemitted by YFP instead of CFP, thus CFP emission is reduced to 80%. If the short-wavelength detector (CFPC) and the long-wavelength detector (YFPC and YFPY) had the same photon sensitivity, a FRET efficiency of 20% would correspond to a FRETr of 20/80 = 0.25. However, in the photometry setup used here, the absolute changes in YFPC were approximately threefold larger than the accompanying changes in CFPC (both in ΔAFU; compare Fig. 2, D with E). A true FRET efficiency of 20% would therefore correspond to a FRETr of 3*20/80 = 0.75. We report FRETr in arbitrary units because the absolute values will differ between setups.

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