Fig 1.
In vitro assay of PLC enzyme activity.
(A) WH-15, a PIP2 analog, is cleaved by PLCs into inositol 1,4,5-triphosphate (IP3), a quinomethide derivative, and 6-aminoquinoline. Fluorescence from 6-aminoquinoline is used to estimate PLC activity. WH-15 has the same inositol as PIP2 that is recognized and cleaved by PLC (dotted rectangle box). (B) Ca2+-dependent activation of PLCβ1. WH-15 (30 μM) was hydrolyzed by recombinant PLCβ1 protein (20 ng) at different free Ca2+ concentrations for 90 min. (C) Real-time PLCβ1 activity was measured with a fixed free Ca2+ concentration at 16.4 μM. Error bars for many points are smaller than the symbol size. n = 3 for each condition.
Fig 2.
Enzyme activity of PLCβ1 in the presence of multivalent cations.
Activity of PLCβ1 was measured and calculated as described in Materials and Methods in the presence of different concentrations of divalent cations (A) and polyamines or neomycin (B). Free Ca2+ concentration was set as 16.4 μM except the measurements using different CaCl2 concentrations. Symbols and lines are experimental data and fitting with our mathematical model, respectively. n = 4–8 for each condition.
Fig 3.
Enzyme activity of PLCγ1 and PLCδ1 in the presence of multivalent cations.
Activity of PLCγ1 (A, B) and PLCδ1 (C, D) was measured as PLCβ1 in Fig 2. Symbols and lines are experimental data and fitting with the mathematical model, respectively. n = 4–8 for PLCγ1 and n = 2–8 for PLCδ1.
Fig 4.
Inhibition of PIP2 hydrolysis by Mg2+ or Ba2+ accumulated into cells through TRPM7.
(A) Schematic diagram. HEK293-TRPM7 cells were transfected with M1R and PH-YFP and TRPM7 channels were activated by voltage clamp to accumulate divalent. (B) Confocal images of PH-YFP where the fluorescence is coded as yellow. Images are taken before and during application of 1 μM oxotremorine-M (Oxo-M) in control group. Top cell was patched. Region of interest (ROI) used for the PH-YFP translocation analysis is indicated in red circle. Black scale bar indicates 20 μm. (C) The average rate of PIP2 hydrolysis by PLC was estimated by the monitoring of translocation of PH-YFP into the cytosol upon activation of M1R with 1 μM Oxo-M. For the accumulation of divalent cations into the cells, external solutions containing 10 mM MgCl2 (N = 5) or 10 mM BaCl2 (N = 7) were perfused at the indicated time, and their influx through TRPM7 was triggered by a negative membrane potential (-80 mV). Error bars were omitted for clarity. (D) Comparison of PLC activity before and after the divalent accumulation. Percent increase of cytosolic PH-YFP upon 2nd Oxo-M treatment was divided by that of 1st Oxo-M (see Methods for details). The results are mean ± SEM and representative of two independent experiments. *** P < 0.001 compared to control group (N = 5).
Fig 5.
Inhibition of PIP2 hydrolysis by Mg2+, spermine, or neomycin dialyzed into cells through patch pipette.
(A; Right) HEK293-tsA201 cells were transfected with M1R and PH-YFP and dialyzed cations through whole-cell patch pipette. (Left) The average rate of PIP2 hydrolysis by PLC was estimated as Fig 1. The first Oxo-M response was triggered while being in cell-attached mode. For accumulation of the cations, the membrane patch was ruptured to form the whole-cell configuration and the cations in the pipette solution were dialyzed into the cell. To accelerate the movement of the cations by electrophoresis, the voltage steps to +80 mV from -80 mV for 2 s were repeated. The effects of 3 mM MgC12 (N = 6), spermine (N = 5), and neomycin (N = 4) are shown. Error bars were omitted for clarity. (B) Summary of PLC activity after pipette perfusion of different concentrations of the cations. PIP2 hydrolysis in the neighboring unpatched cells was nearly unaltered. The results are mean ± SEM and representative of three independent experiments. N = 4–10 for each condition. ** P < 0.01 and *** P < 0.001 compared to 1 mM MgCl2 group (N = 10).
Fig 6.
Expression of PLCβ in HEK293-tsA201 and HEK293-TRPM7 cells.
Expression level of PLCβ isoforms was analyzed with Q-PCR. Their mRNA levels were normalized to GAPDH and presented as relative to PLCβ1. n = 3 for each condition. * P < 0.01, ** P < 0.01, and *** P < 0.001 compared to PLCβ1 group. # P < 0.05 and ### P < 0.001 compared to PLCβ2 group.
Fig 7.
Working model for the modulation of PLCs activity via charge shielding of PIP2 by cations.
(Left) After activation of PLC by Gq-coupled GPCRs or receptor tyrosine kinases as indicated by lightning flash, PLC hydrolyzes PIP2 and generates intracellular second messengers, IP3 and DAG. (Middle) Positively charged divalent cations or polyamines accumulate around the negatively charged PIP2 and reduce electrostatic interaction between PIP2 and PLC, resulting less PIP2 hydrolysis. (Right) Highly charged neomycin inhibits PLC similarly. However the charge shielding by neomycin does not inhibit the PLCs activity completely, possibly due to their limited accumulation around PIP2 which, in turn, is caused by either too large size or too high charge density. See text for details.