Gbetagamma-dependent and Gbetagamma-independent basal activity of G protein-activated K+ channels. Export

@article{citeulike:4409316, abstract = {Cardiac and neuronal G protein-activated K+ channels (GIRK; Kir3) open following the binding of Gbetagamma subunits, released from Gi/o proteins activated by neurotransmitters. GIRKs also possess basal activity contributing to the resting potential in neurons. It appears to depend largely on free Gbetagamma, but a Gbetagamma-independent component has also been envisaged. We investigated Gbetagamma dependence of the basal GIRK activity (A(GIRK,basal)) quantitatively, by titrated expression of Gbetagamma scavengers, in Xenopus oocytes expressing GIRK1/2 channels and muscarinic m2 receptors. The widely used Gbetagamma scavenger, myristoylated C terminus of beta-adrenergic kinase (m-cbetaARK), reduced A(GIRK,basal) by 70-80\% and eliminated the acetylcholine-evoked current (I(ACh)). However, we found that m-cbetaARK directly binds to GIRK, complicating the interpretation of physiological data. Among several newly constructed Gbetagamma scavengers, phosducin with an added myristoylation signal (m-phosducin) was most efficient in reducing GIRK currents. m-phosducin relocated to the membrane fraction and did not bind GIRK. Titrated expression of m-phosducin caused a reduction of A(GIRK,basal) by up to 90\%. Expression of GIRK was accompanied by an increase in the level of Gbetagamma and Galpha in the plasma membrane, supporting the existence of preformed complexes of GIRK with G protein subunits. Increased expression of Gbetagamma and its constitutive association with GIRK may underlie the excessively high A(GIRK,basal) observed at high expression levels of GIRK. Only 10-15\% of A(GIRK,basal) persisted upon expression of both m-phosducin and cbetaARK. These results demonstrate that a major part of Ibasal is Gbetagamma-dependent at all levels of channel expression, and only a small fraction (<10\%) may be Gbetagamma-independent.}, author = {Rishal, I. and Porozov, Y. and Yakubovich, D. and Varon, D. and Dascal, N.}, citeulike-article-id = {4409316}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M412196200}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/15728579}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=15728579}, day = {29}, doi = {10.1074/jbc.M412196200}, issn = {0021-9258}, journal = {The Journal of biological chemistry}, keywords = {girk}, month = {April}, number = {17}, pages = {16685--16694}, posted-at = {2009-04-27 07:07:26}, priority = {3}, title = {Gbetagamma-dependent and Gbetagamma-independent basal activity of G protein-activated K+ channels.}, url = {http://dx.doi.org/10.1074/jbc.M412196200}, volume = {280}, year = {2005} }

TY - JOUR ID - citeulike:4409316 L3 - citeulike-article-id:4409316 N2 - Cardiac and neuronal G protein-activated K+ channels (GIRK; Kir3) open following the binding of Gbetagamma subunits, released from Gi/o proteins activated by neurotransmitters. GIRKs also possess basal activity contributing to the resting potential in neurons. It appears to depend largely on free Gbetagamma, but a Gbetagamma-independent component has also been envisaged. We investigated Gbetagamma dependence of the basal GIRK activity (A(GIRK,basal)) quantitatively, by titrated expression of Gbetagamma scavengers, in Xenopus oocytes expressing GIRK1/2 channels and muscarinic m2 receptors. The widely used Gbetagamma scavenger, myristoylated C terminus of beta-adrenergic kinase (m-cbetaARK), reduced A(GIRK,basal) by 70-80% and eliminated the acetylcholine-evoked current (I(ACh)). However, we found that m-cbetaARK directly binds to GIRK, complicating the interpretation of physiological data. Among several newly constructed Gbetagamma scavengers, phosducin with an added myristoylation signal (m-phosducin) was most efficient in reducing GIRK currents. m-phosducin relocated to the membrane fraction and did not bind GIRK. Titrated expression of m-phosducin caused a reduction of A(GIRK,basal) by up to 90%. Expression of GIRK was accompanied by an increase in the level of Gbetagamma and Galpha in the plasma membrane, supporting the existence of preformed complexes of GIRK with G protein subunits. Increased expression of Gbetagamma and its constitutive association with GIRK may underlie the excessively high A(GIRK,basal) observed at high expression levels of GIRK. Only 10-15% of A(GIRK,basal) persisted upon expression of both m-phosducin and cbetaARK. These results demonstrate that a major part of Ibasal is Gbetagamma-dependent at all levels of channel expression, and only a small fraction (<10%) may be Gbetagamma-independent. IS - 17 JF - The Journal of biological chemistry SN - 0021-9258 EP - 16694 TI - Gbetagamma-dependent and Gbetagamma-independent basal activity of G protein-activated K+ channels. VL - 280 SP - 16685 KW - girk AU - Rishal, I AU - Porozov, Y AU - Yakubovich, D AU - Varon, D AU - Dascal, N PY - 2005/04/29/ UR - http://dx.doi.org/10.1074/jbc.M412196200 ER -