Abstract
The patch-clamp technique was employed to investigate phosphorylation/dephosphorylation-dependent modulation of L-type Ca2+ channels in smooth-muscle cells isolated from human umbilical vein. Okadaic acid, an inhibitor of phosphoprotein phosphatases type 1 (PP1) and 2A (PP2A), increased the probability of channels being in the open state (Po) in intact cells. This increase in Po was due mainly to promotion of long-lasting channel openings, i.e. promotion of 'mode 2' gating behaviour. Exposure of the cytoplasmic side of excised patches of membrane to the purified catalytic subunit of PP2A (PP2Ac) resulted in the opposite modulation of channel function. PP2Ac (0.2 unit/ml) reduced the Po of Ca2+ channels mainly via suppression of 'mode 2' gating. This effect of PP2Ac was completely prevented by 1 microM okadaic acid. The catalytic subunit of PPI (0.2 unit/ml), however, barely affected channel activity. Our results provide evidence for a PP2A-sensitive regulatory site that controls modal gating of L-type Ca2+ channels in smooth muscle.
Full Text
The Full Text of this article is available as a PDF (455.6 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Braulke T., Mieskes G. Role of protein phosphatases in insulin-like growth factor II (IGF II)-stimulated mannose 6-phosphate/IGF II receptor redistribution. J Biol Chem. 1992 Aug 25;267(24):17347–17353. [PubMed] [Google Scholar]
- Curtis B. M., Catterall W. A. Phosphorylation of the calcium antagonist receptor of the voltage-sensitive calcium channel by cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2528–2532. doi: 10.1073/pnas.82.8.2528. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Groschner K., Schuhmann K., Baumgartner W., Pastushenko V., Schindler H., Romanin C. Basal dephosphorylation controls slow gating of L-type Ca2+ channels in human vascular smooth muscle. FEBS Lett. 1995 Oct 2;373(1):30–34. doi: 10.1016/0014-5793(95)01012-4. [DOI] [PubMed] [Google Scholar]
- Hescheler J., Mieskes G., Rüegg J. C., Takai A., Trautwein W. Effects of a protein phosphatase inhibitor, okadaic acid, on membrane currents of isolated guinea-pig cardiac myocytes. Pflugers Arch. 1988 Aug;412(3):248–252. doi: 10.1007/BF00582504. [DOI] [PubMed] [Google Scholar]
- Jahn H., Nastainczyk W., Röhrkasten A., Schneider T., Hofmann F. Site-specific phosphorylation of the purified receptor for calcium-channel blockers by cAMP- and cGMP-dependent protein kinases, protein kinase C, calmodulin-dependent protein kinase II and casein kinase II. Eur J Biochem. 1988 Dec 15;178(2):535–542. doi: 10.1111/j.1432-1033.1988.tb14480.x. [DOI] [PubMed] [Google Scholar]
- Lai Y., Peterson B. Z., Catterall W. A. Selective dephosphorylation of the subunits of skeletal muscle calcium channels by purified phosphoprotein phosphatases. J Neurochem. 1993 Oct;61(4):1333–1339. doi: 10.1111/j.1471-4159.1993.tb13626.x. [DOI] [PubMed] [Google Scholar]
- O'Callahan C. M., Hosey M. M. Multiple phosphorylation sites in the 165-kilodalton peptide associated with dihydropyridine-sensitive calcium channels. Biochemistry. 1988 Aug 9;27(16):6071–6077. doi: 10.1021/bi00416a036. [DOI] [PubMed] [Google Scholar]
- Obara K., Yabu H. Dual effect of phosphatase inhibitors on calcium channels in intestinal smooth muscle cells. Am J Physiol. 1993 Feb;264(2 Pt 1):C296–C301. doi: 10.1152/ajpcell.1993.264.2.C296. [DOI] [PubMed] [Google Scholar]
- Ochi R., Kawashima Y. Modulation of slow gating process of calcium channels by isoprenaline in guinea-pig ventricular cells. J Physiol. 1990 May;424:187–204. doi: 10.1113/jphysiol.1990.sp018062. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ono K., Fozzard H. A. Two phosphatase sites on the Ca2+ channel affecting different kinetic functions. J Physiol. 1993 Oct;470:73–84. doi: 10.1113/jphysiol.1993.sp019848. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Romanin C., Grösswagen P., Schindler H. Calpastatin and nucleotides stabilize cardiac calcium channel activity in excised patches. Pflugers Arch. 1991 Mar;418(1-2):86–92. doi: 10.1007/BF00370456. [DOI] [PubMed] [Google Scholar]
- Schmid R., Seydl K., Baumgartner W., Groschner K., Romanin C. Trypsin increases availability and open probability of cardiac L-type Ca2+ channels without affecting inactivation induced by Ca2+. Biophys J. 1995 Nov;69(5):1847–1857. doi: 10.1016/S0006-3495(95)80055-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schuhmann K., Groschner K. Protein kinase-C mediates dual modulation of L-type Ca2+ channels in human vascular smooth muscle. FEBS Lett. 1994 Mar 21;341(2-3):208–212. doi: 10.1016/0014-5793(94)80458-3. [DOI] [PubMed] [Google Scholar]
- Takai A., Mieskes G. Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases. Biochem J. 1991 Apr 1;275(Pt 1):233–239. doi: 10.1042/bj2750233. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsien R. W., Bean B. P., Hess P., Lansman J. B., Nilius B., Nowycky M. C. Mechanisms of calcium channel modulation by beta-adrenergic agents and dihydropyridine calcium agonists. J Mol Cell Cardiol. 1986 Jul;18(7):691–710. doi: 10.1016/s0022-2828(86)80941-5. [DOI] [PubMed] [Google Scholar]
- Wiechen K., Yue D. T., Herzig S. Two distinct functional effects of protein phosphatase inhibitors on guinea-pig cardiac L-type Ca2+ channels. J Physiol. 1995 May 1;484(Pt 3):583–592. doi: 10.1113/jphysiol.1995.sp020688. [DOI] [PMC free article] [PubMed] [Google Scholar]