Mouse aortic smooth muscle cells (SMCs) were loaded for 72 h with cholesterol by using cholesterol:methyl-␤-cyclodextrin complexes, leading to Ϸ2-fold and Ϸ10-fold increases in the contents of total cholesterol and cholesteryl ester, respectively. Foam-cell formation was demonstrated by accumulation of intracellular, Oil Red O-stained lipid droplets. Immunostaining showed decreased protein levels of smooth muscle ␣-actin and ␣-tropomyosin and increased levels of macrophage markers CD68 and Mac-2 antigen. Quantitative real-time RT-PCR revealed that after cholesterol loading, the expression of SMC-related genes ␣-actin, ␣-tropomyosin, myosin heavy chain, and calponin H1 decreased (to 11.5 ؎ 0.5%, 29.3 ؎ 1.4%, 23.8 ؎ 1.4%, and 3.8 ؎ 0.5% of unloaded cells, respectively; P < 0.05 for all), whereas expression of macrophage-related genes CD68, Mac-2, and ABCA1 mRNA increased (to 709 ؎ 84%, 330 ؎ 11%, and 207 ؎ 13% of unloaded cells, respectively; P < 0.05 for all), thereby demonstrating that the protein changes were regulated at the mRNA level.

Voltage-gated Kv7 (KCNQ) channels underlie important K ϩ currents, including the neuronal M current, and are thought to be sensitive to membrane phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and PIP 2 depletion to underlie muscarinic receptor inhibition. We studied regulation of Kv7.2-7.4 channels by PIP 2 in Chinese hamster ovary (

Modulation of voltage-gated Ca2ϩ channels via G-protein-coupled receptors is a prime mechanism regulating neurotransmitter release and synaptic plasticity. Despite extensive studies, the molecular mechanism underlying G q/11 -mediated modulation remains unclear. We found cloned and native N-type Ca 2ϩ channels to be regulated by phosphotidylinositol 4,5-bisphosphate (PIP 2 ). In inside-out oocyte patches, PIP 2 greatly attenuated or reversed the observed rundown of expressed channels. In sympathetic neurons, muscarinic M 1 ACh receptor suppression of the Ca 2ϩ current (I Ca ) was temporally correlated with PIP 2 hydrolysis, blunted by PIP 2 in whole-cell pipettes, attenuated by expression of PIP 2 -sequestering proteins, and became irreversible when PIP 2 synthesis was blocked. We also probed mechanisms of receptor specificity. Although bradykinin also induced PIP 2 hydrolysis, it did not inhibit I Ca . However, bradykinin receptors became nearly as effective as M 1 receptors when PIP 2 synthesis, IP 3 receptors, or the activity of neuronal Ca 2ϩ sensor-1 were blocked, suggesting that bradykinin receptor-induced intracellular Ca 2ϩ increases stimulate PIP 2 synthesis, compensating for PIP 2 hydrolysis. We suggest that differential use of PIP 2 signals underlies specificity of G q/11 -coupled receptor actions on the channels.

To quantify the modulation of KCNQ2/3 current by [Ca2+]i and to test if calmodulin (CaM) mediates this action, simultaneous whole-cell recording and Ca2+ imaging was performed on CHO cells expressing KCNQ2/3 channels, either alone, or together with wild-type (wt) CaM, or dominant-negative (DN) CaM. We varied [Ca2+]i from <10 to >400 nM with ionomycin (5 μM) added to either a 2 mM Ca2+, or EGTA-buffered Ca2+-free, solution. Coexpression of wt CaM made KCNQ2/3 currents highly sensitive to [Ca2+]i (IC50 70 ± 20 nM, max inhibition 73%, n = 10). However, coexpression of DN CaM rendered KCNQ2/3 currents largely [Ca2+]i insensitive (max inhibition 8 ± 3%, n = 10). In cells without cotransfected CaM, the Ca2+ sensitivity was variable but generally weak. [Ca2+]i modulation of M current in superior cervical ganglion (SCG) neurons followed the same pattern as in CHO cells expressed with KCNQ2/3 and wt CaM, suggesting that endogenous M current is also highly sensitive to [Ca2+]i. Coimmunoprecipitations showed binding of CaM to KCNQ2–5 that was similar in the presence of 5 mM Ca2+ or 5 mM EGTA. Gel-shift analyses suggested Ca2+-dependent CaM binding to an “IQ-like” motif present in the carboxy terminus of KCNQ2–5. We tested whether bradykinin modulation of M current in SCG neurons uses CaM. Wt or DN CaM was exogenously expressed in SCG cells using pseudovirions or the biolistic “gene gun.” Using both methods, expression of both wt CaM and DN CaM strongly reduced bradykinin inhibition of M current, but for all groups muscarinic inhibition was unaffected. Cells expressed with wt CaM had strongly reduced tonic current amplitudes as well. We observed similar [Ca2+]i rises by bradykinin in all the groups of cells, indicating that CaM did not affect Ca2+ release from stores. We conclude that M-type currents are highly sensitive to [Ca2+]i and that calmodulin acts as their Ca2+ sensor.