Localization and Dynamics of Phosphatidylinositol 4,5-Bisphosphate (PIP2) in Adult Skeletal Muscle Fibers

Abstract

PIP2 is a precursor of important second messengers, and by itself is a direct modulator of the activity of ion channels and transporters. We investigated the localization and dynamic changes in PIP2 levels in live adult muscle fibers by expressing two PIP2 sensors: an EGFP construct of the pleckstrin homology (PH) domain of the phospholipase delta1 subunit (PH-EGFP), and a construct of the PIP2 binding domain of the tubby protein (EGFP-tubby). EGFP-tubby has higher affinity and specificity for PIP2 than PH-EGFP, and has been used to sequester PIP2 in the plasma membrane. Their respective plasmids (∗) were transfected by in vivo electroporation of FDB muscles. Two-photon laser scanning microscopy (TPLSM) shows that both EGFP-tubby and PH-EGFP are efficiently expressed in muscle fibers and that they are distributed in a double-banded pattern indicating localization at the transverse tubular system (TTS) membranes (in addition to the sarcolemma). Peak/baseline ratio analysis of TPLSM images suggests that there is a larger proportion of EGFP-tubby than PH-EGFP associated with the TTS membranes. Although the expression of PH-EGFP does not change neither the voltage-dependence nor the amplitude of Ca2+ release signals detected with Rhod-5N, the expression of EGFP-tubby apparently left-shifts their voltage-dependence by >10 mV. To further investigate the localization and dynamics of PIP2 in the TTS, we performed fluorescence resonance energy transfer (FRET) studies. The voltage-dependence of FRET signals, based on the translocation of the lipophilic anion dipicrylamine (DPA), shows that the EGFP tags of both PH-EGFP and EGFP-tubby are anchored within ∼6-9 nm of the TTS membrane. (∗)The plasmids were kindly provided to us by Dr. Tamas Balla, NICHD, NIH. This work was supported by NIH grants AR047664, AR041802, and AR054816.