Regulation of stably expressed and native BK channels from human myometrium by cGMP- and cAMP-dependent protein kinase.
The cloned BK channel alpha subunit from human myometrium was stably expressed in Chinese hamster ovary cells, either alone (CHOalpha cells) or in combination with the auxiliary beta subunit (CHOalpha+beta cells). We studied basic channel properties and the effects of cGMP- and cAMP-dependent protein kinases on the BK channel activity. Coexpression of alpha and beta subunits enhanced the Ca2+ and voltage sensitivity of the BK channel, and decreased the inhibitory potency of iberiotoxin. Blocking and stimulating effects on BK channel activity by charybdotoxin and nitric oxide, respectively, were independent of the beta subunit. The cGMP kinase Ialpha and cAMP kinase failed to affect BK channel activity in CHOalpha and CHOalpha+beta cells at different [Ca2+]i and voltages. In contrast, BK channels in freshly isolated myometrial cells from postmenopausal women responded to cAMP kinase and cGMP kinase with a fourfold and twofold decrease in their open probability (NPo), respectively. These effects could be reversed by alkaline phosphatase and remained unaffected by the phosphatase inhibitor okadaic acid (100 nM). In 28% of myometrial cells, however, cAMP and cGMP kinases increased NPo 2-fold and 3.5-fold, respectively. This stimulation was enhanced rather than reversed by alkaline phosphatase and was abolished by 100 nM okadaic acid. The results suggest that in stably transfected CHO cells the expressed BK channel is not regulated by cAMP kinase and cGMP kinase. However, in native myometrial cells stimulatory and inhibitory regulation of BK channels by cAMP kinase and cGMP kinase was observed, suggesting that channel regulation by the protein kinases requires factors that are not provided by CHO cells. Alternatively, failure of regulation may have been due to the primary structure of the myometrial BK channel protein used in this study.
Institut für Pharmakologie und Toxikologie der Technischen Universität München, Biedersteiner Strasse 29, D-80802 Munich, Germany.
X B
XB
Zhou
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J
Schlossmann
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F
Hofmann
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P
Ruth
M
M
Korth
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D018999:Charybdotoxin / Q000494:pharmacology; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology*; D005260:Female; D015870:Gene Expression; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008564:Membrane Potentials / Q000187:drug effects; D009215:Myometrium / Q000166:cytology* / Q000187:drug effects; D009569:Nitric Oxide / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000096:biosynthesis* / Q000235:genetics; D024681:Potassium Channels, Calcium-Activated; D014162:Transfection
D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D010455:Peptides; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D018999:Charybdotoxin; D009569:Nitric Oxide; C064719:iberiotoxin; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D011188:Potassium; D002118:Calcium