Molecular determinants of ATP-sensitive potassium channel MgATPase activity: diabetes risk variants and diazoxide sensitivity

Molecular interactions between two residues in the sulfonylurea receptor (SUR) subunit of the ATP-sensitive potassium channel influence MgATPase activity. This interaction may provide a mechanism for the increased diabetes risk associated with a common channel variant and determines sensitivity to diazoxide.

Estrogen Modulation of MgATPase Activity of Nonmuscle Myosin-II-B Filaments

The study tested the hypothesis that estrogen controls epithelial paracellular resistance through modulation of myosin. The objective was to understand how estrogen modulates nonmuscle myosin-II-B (NMM-II-B), the main component of the cortical actomyosin in human epithelial cervical cells. Experiments used human cervical epithelial cells CaSki as a model, and end points were NMM-II-B phosphorylation, filamentation, and MgATPase activity. The results were as follows: 1) treatment with estrogen increased phosphorylation and MgATPase activity and decreased NMM-II-B filamentation; 2) estrogen effects could be blocked by antisense nucleotides for the estrogen receptor-α and by ICI-182,780, tamoxifen, and the casein kinase-II (CK2) inhibitor, 5,6-dichloro-1-β-(D)-ribofuranosylbenzimidazole and attenuated by AG1478 and PD98059 (inhibitors of epithelial growth factor receptor and ERK/MAPK) but not staurosporine [blocker of protein kinase C (PKC)]; 3) treatments with the PKC activator sn-1,2-dioctanoyl diglyceride induced biphasic effect on NMM-II-B MgATPase activity: an increase at 1 nm to 1 μm and a decrease in activity at more than 1 μm; 4) sn-1,2-dioctanoyl diglyceride also decreased NMM-II-B filamentation in a monophasic and saturable dose dependence (EC50 1–10 μm); 5) when coincubated directly with purified NMM-II-B filaments, both CK2 and PKC decreased filamentation and increased MgATPase activity; 6) assays done on disassembled NMM-II-B filaments showed MgATPase activity in filaments obtained from estrogen-treated cells but not estrogen-depleted cells; and 7) incubations in vitro with CK2, but not PKC, facilitated MgATPase activity, even in disassembled NMM-II-B filaments. The results suggest that estrogen, in an effect mediated by estrogen receptor-α and CK2 and involving the epithelial growth factor receptor and ERK/MAPK cascades, increases NMM-II-B MgATPase activity independent of NMM-II-B filamentation status.

Cardioprotective effects of exercise training on myofilament calcium activation in ovariectomized rats

The risks associated with hormone replacement therapy, especially cardiac diseases in postmenopausal women, have prompted extensive studies for other preventive or therapeutic alternatives. We investigated the cardioprotective effects of exercise training on the changes in cardiac myofilament Ca2+ activation in 10-wk-old ovariectomized rats. The exercise groups were subjected to a 9-wk running program on a motor-driven treadmill 1 wk after surgery. The relationship between pCa (-log molar free Ca2+ concentration) and myofibrillar MgATPase activity of exercise-sham myofibrils or exercise-ovariectomized myofibrils was the same and could not be distinguished from that of sedentary-sham control hearts. In contrast, a significant suppression in maximum MgATPase activity and a leftward shift of pCa50 (half-maximally activating pCa) in the pCa-ATPase activity relationship were detected in sedentary-ovariectomized rats. Exercise training also prevented the shift in myosin heavy chain (MHC) isoforms toward β-MHC in ovariectomized hearts. The upregulation of β1-adrenergic receptors in the left ventricular membranes of ovariectomized rat hearts, as measured by receptor binding and immunoblot analyses, was no longer observed in exercise-ovariectomized hearts. Immunoblot analyses of heat shock protein (HSP) 72, an inducible form of HSP70, demonstrated a significant downregulation in ovariectomized hearts. Exercise training in ovariectomized rats completely reversed the expression of HSP72 to the same level as sham controls. Our results clearly indicate the cardioprotective effects of exercise training on changes in cardiac myofilament Ca2+ activation in ovariectomized rats. Alterations in expression of β1-adrenergic receptors and HSP72 may, in part, play a mechanistic role in the cardioprotective effects.

Troponin I serines 43/45 and regulation of cardiac myofilament function

We studied Ca2+ dependence of tension and actomyosin ATPase rate in detergent extracted fiber bundles isolated from transgenic mice (TG), in which cardiac troponin I (cTnI) serines 43 and 45 were mutated to alanines (cTnI S43A/S45A). Basal phosphorylation levels of cTnI were lower in TG than in wild-type (WT) mice, but phosphorylation of cardiac troponin T was increased. Compared with WT, TG fiber bundles showed a 13% decrease in maximum tension and a 20% increase in maximum MgATPase activity, yielding an increase in tension cost. Protein kinase C (PKC) activation with endothelin (ET) or phenylephrine plus propranolol (PP) before detergent extraction induced a decrease in maximum tension and MgATPase activity in WT fibers, whereas ET or PP increased maximum tension and stiffness in TG fibers. TG MgATPase activity was unchanged by ET but increased by PP. Measurement of protein phosphorylation revealed differential effects of agonists between WT and TG myofilaments and within the TG myofilaments. Our results demonstrate the importance of PKC-mediated phosphorylation of cTnI S43/S45 in the control of myofilament activation and cross-bridge cycling rate.