Abstract-Many cells are constantly exposed to fluid mechanical forces generated by flowing blood, and wall shear stresses modulate aspects of their structure and function. However, the mechanisms for mechanotransduction of flow are not well understood. Here we report that TRPM7, which is both an ion channel and a functional kinase, is translocated within cells in response to laminar flow. After exposure of cells to physiological values of laminar fluid flow, the number of TRPM7 molecules localized at or near the plasma membrane increased up to 2-fold, in less than 100 seconds. This increase in membrane-localized GFP-TRPM7, as seen by total internal reflection fluorescence microscopy, closely correlated with increases in TRPM7 current. Both endogenous and heterologously expressed TRPM7 was found in tubulovesicular structures that were translocated to the region of the plasma membrane on induction of shear stress. In vascular smooth muscle cells, but not in several types of endothelial cells, fluid flow increased endogenous native TRPM7 current amplitude. We hypothesize that TRPM7 plays a role in pathological response to vessel wall injury. Key Words: TRP ion channels Ⅲ TRPM7 Ⅲ shear force Ⅲ total internal reflection fluorescence (TIRF) microscopy Ⅲ vascular smooth muscle cells T RP ion channels are often found in sensory systems, but their wide distribution suggests they are cellular sensors in a broad sense. 1 TRPM6 and TRPM7 are unique channels that possess both ion channel and protein kinase activities. The C-terminal kinase domain bears little sequence identity with other kinases but is enzymatically active and structurally homologous to protein kinase A. 2,3 The physiological mechanism for activation of the widely expressed TRPM7 is unknown. TRPM7 channels are inhibited by phospholipase C (PLC)-catalyzed phosphatidylinositol 4,5-bisphosphate (PIP 2 ) hydrolysis 4 and reportedly regulated through protein kinase A by receptors coupled to adenylyl cyclase. 5 TRPM7 may be involved in anoxia-induced cell death in brain 6 and has been reported to be required for cell viability in TRPM7-null avian DT40 cells. 7 Cells sense shear stress-induced mechanical stimulation and convert it into a biochemical response that impacts normal and abnormal tissue development, including growth, differentiation, migration, gene expression, protein synthesis, and apoptosis. 8 The response of cells to fluid flow depends on the cell type and the magnitude and characteristics of the shear stress applied under physiological or pathological conditions. Endothelial cells are constantly subjected to blood flow that regulates physiological blood vessel responses as well as pathological arterial wall responses. In large arteries, the endothelium is exposed to shear stress values in the range 10 to 40 dyne/cm 2 . 9 The vascular smooth muscle cells are protected from shear stress by endothelial cell lining under physiological conditions but become exposed to shear stress after endothelial injury.Ion channels can respond to shear stress. 10,11 A...

Low-voltage-activated (LVA) T-type calcium channels have a wide tissue distribution and have well-documented roles in the control of action potential burst generation and hormone secretion. In neurons of the central nervous system and secretory cells of the adrenal and pituitary, LVA channels are inhibited by activation of G-protein-coupled receptors that generate membrane-delimited signals, yet these signals have not been identified. Here we show that the inhibition of alpha1H (Ca(v)3.2), but not alpha(1G) (Ca(v)3.1) LVA Ca2+ channels is mediated selectively by beta2gamma2 subunits that bind to the intracellular loop connecting channel transmembrane domains II and III. This region of the alpha1H channel is crucial for inhibition, because its replacement abrogates inhibition and its transfer to non-modulated alpha1G channels confers beta2gamma2-dependent inhibition. betagamma reduces channel activity independent of voltage, a mechanism distinct from the established betagamma-dependent inhibition of non-L-type high-voltage-activated channels of the Ca(v)2 family. These studies identify the alpha1H channel as a new effector for G-protein betagamma subunits, and highlight the selective signalling roles available for particular betagamma combinations.

To assess the relation between usual nutrient intake and subsequently diagnosed age-related nuclear lens opacities.Subjects: Four hundred seventy-eight nondiabetic women aged 53 to 73 years from the Boston, Mass, area without previously diagnosed cataracts sampled from the Nurses' Health Study cohort.Methods: Usual nutrient intake was calculated as the average intake from 5 food frequency questionnaires that were collected during a 13-to 15-year period before the evaluation of lens opacities. The duration of vitamin supplement use was determined from 7 questionnaires collected during this same period. We defined nuclear opacities as a nuclear opalescence grade of 2.5 or higher using the Lens Opacification Classification System III. Results:The prevalence of nuclear opacification was significantly lower in the highest nutrient intake quintile category relative to the lowest quintile category for vitamin C (PϽ.001), vitamin E (P = .02), riboflavin (P=.005), folate (P=.009), ␤-carotene (P=.04), and lutein/ zeaxanthin (P = .03). After adjustment for other nutrients, only vitamin C intake remained significantly associated (P=.003 for trend) with the prevalence of nuclear

Low-voltage-activated (LVA) Ca2+ channels are widely distributed throughout the CNS and are important determinants of neuronal excitability, initiating dendritic and somatic Ca2+ spikes that trigger and shape the pattern of action potential firing. Here, we define a molecular mechanism underlying the dynamic regulation of alpha1H channels (Cav3.2), by Ca2+/CaM-dependent protein kinase II (CaMKII). We show that channel regulation is selective for the LVA alpha1H Ca2+ channel subtype, depends on determinants in the alpha1H II-III intracellular loop, and requires the phosphorylation of a serine residue absent from unregulated alpha1G (Cav3.1) channels. These studies identify the alpha1H channel as a new substrate for CaMKII and provide the first molecular mechanism for the direct regulation of T-type Ca2+ channels by a protein kinase. Our data suggest a novel mechanism for modulating the integrative properties of neurons.