Martin Mayrleitner

The calcium release channel of sarcoplasmic reticulum is modulated by FK-506 binding protein : effect of FKBP-12 on single channel activity of the skeletal muscle ryanodine receptor

The calcium release channel/ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum is tightly associated with the immunophilin FK-506 binding protein (FKBP-12). The immunosuppressant drug FK-506 effectively dissociates FKBP-12 from the calcium release channel of terminal cisternae (TC) vesicles. Furthermore, calcium flux measurements of TC indicate that FKBP-12 stabilizes the closed conformation of the calcium release channel of TC [Timerman AP, Ogunbunmi E, Freund EA, Wiederrecht G, Marks AM, Fleischer S. (1993) J. Biol. Chem., 268, 22992-22999]. In this report, the effect of FKBP on single channel recordings of the calcium release channel/ryanodine receptor of TC is measured directly. Single channel recordings of the ryanodine receptor were obtained by fusion of TC vesicles into planar bilayers. The channel devoid of FKBP, retains key diagnostic features. That is, activation by Ca2+ and ryanodine, inhibition by Mg2+ (mM) and ruthenium red (microM), and its unitary conductance remain the same. Recordings of the calcium release channel obtained from the FKBP-deficient TC vesicles, as compared with control TC, have greater open probability and longer mean open times in a free calcium concentration range of 70 nM to 1.2 microM. The sensitivity of the channel to caffeine is also enhanced by the removal of FKBP. The enhanced channel activation of FKBP-deficient TC is reversed by rebinding recombinant FKBP-12 in a cyclical fashion. We conclude that FKBP modifies the channel behavior of the calcium release channel of skeletal muscle sarcoplasmic reticulum.

IP3 receptor purified from liver plasma membrane is an (1,4,5)IP3 activated and (1,3,4,5)IP4 inhibited calcium permeable ion channel

The IP3 receptor is involved in Ca2+ mobilization from intracellular stores. Recently, we purified an inositol (1,4,5)-trisphosphate receptor from rat liver plasma membrane (LPM-IP3R) [Schäfer R. Hell K. Fleischer S. (1993) Purification of an IP3 receptor from liver plasma membrane. Biophys. J. 66, A146]. The purified LPM-IP3 receptor was incorporated into vesicle derived planar bilayers and its channel properties characterized. The receptor displayed ion channel activity that was activated by inositol (1,4,5)-trisphosphate [(1,4,5)IP3] (1 microM) and inhibited by inositol (1,3,4,5)-tetrakisphosphate (IC50 approximately 1 microM) and by heparin (IC50 approximately 20 micrograms/ml). The channel displays a unitary conductance of 9 pS, and 13 pS in symmetrical 100 mM and 500 mM KCl, respectively, and in symmetrical 250 mM cesium methanesulfonate the slope conductance is 11 pS. Activation by (1,4,5)IP3 is specific to the cis-side of the chamber, equivalent to the cytoplasmic face. The receptor is a Ca2+ permeable ion channel based on ion selectivity (Ca2+ > K+ > Na+ >> Cl). The LPM-IP3 receptor was also permeable to Cs (Cs+ > or = K+), similar to other intracellular Ca2+ release channels, i.e. the IP3 receptor from brain and smooth muscle (IP3R-1) and the ryanodine receptor from skeletal muscle (RyR-1) and heart (RyR-2). Channel activity is not voltage dependent (+/- 100 mV applied voltage). The channel is activated by ATP and Ca2+. The open probability of the (1,4,5)IP3 activated channel activity displays a bell shaped response to cis Ca2+ ion concentration of our system. The LPM-IP3 receptor differs from intracellular IP3R-1 in that the Ca2+ and ATP concentration required for maximum activation is about 10 times higher as compared with IP3R-1 from brain cerebellum and smooth muscle. We conclude that the LPM-IP3 receptor is an (1,4,5)IP3 activated Ca2+ permeable ion channel. The implication of our studies is that in liver, (1,4,5)IP3 regulates Ca2+ influx via the plasma membrane.

Phosphorylation with protein kinases modulates calcium loading of terminal cisternae of sarcoplasmic reticulum from skeletal muscle

We previously found in single channel studies that ryanodine receptor (RyR) channel activity can be made insensitive to block by Mg2+ when terminal cisternae of sarcoplasmic reticulum, incorporated into planar bilayers, are treated with protein kinase A (PKA) or Ca2+/calmodulin dependent protein kinase type II (CamPK II), and then again made sensitive by treatment with protein phosphatases [Hain J. Nath S. Mayrleitner M. Fleischer S. Schindler H. (1994) Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. Biophys. J., 67, 1823-1833]. In this study, modulation by protein kinases and phosphatases on net Ca2+ uptake by TC is presented. Phosphorylation of TC vesicles with PKA, CamPK II, or protein kinase C (PKC) reduced the calcium loading rate of TC vesicles 3-fold, 2.1-fold and 1.7-fold, respectively, measured in the presence of 1 mM MgCl2. There is no effect when AMP-PNP is substituted for ATP. Phosphorylation of the RyR was also measured by incorporation of [gamma-32P]-phosphate from ATP. A phosphorylation stoichiometry of 1.94 +/- 0.1 (32P/RyR) for PKA, 0.89 +/- 0.08 for CamPK II and 0.95 +/- 0.16 for PKC was obtained under these conditions. A study of the time dependence of phosphorylation with PKA and CamPK shows a direct correlation of reduction in calcium loading rate with increased phosphorylation of the ryanodine receptor. Treatment with protein phosphatase 1 enhanced the calcium loading rate again, after it was reduced by PKA phosphorylation. Investigation of the magnesium dependency shows that even at higher [Mg2+] (6 mM), PKA phosphorylated TC vesicles have a 2.3-fold reduced calcium loading rate indicating insensitivity to block by Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)