Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when coexpressed with CaM and CaMΔN. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ΔEF-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.
Binding Sites ; Calcium ; Calcium Signaling ; Calmodulin ; Ion Channels ; Ions ; Patch-Clamp Techniques ; Polycystic Kidney Diseases ; Transient Receptor Potential Channels

Gα(q)-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)P₂) depletion. When PI(4,5)P₂ depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gα(q)-phospholipase C β (Gα(q)-PLCβ) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCβ activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca²⁺ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca²⁺ due to Ca²⁺ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)P₂ depletion.
Calcium ; Cytoplasm ; Endoplasmic Reticulum ; GTP-Binding Proteins ; HEK293 Cells ; Inositol ; Phosphatidylinositol 4,5-Diphosphate ; Phospholipases ; Phosphotransferases ; Protein Kinase C ; Transient Receptor Potential Channels ; Type C Phospholipases

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. It is suggested that TRPC4β is regulated by phospholipase C (PLC) signaling and is especially maintained by phosphatidylinositol 4,5-bisphosphate (PIP2 ). In this study, we present the regulation mechanism of the TRPC4 channel with PIP2 hydrolysis which is mediated by a channel-bound PLCδ1 but not by the GqPCR signaling pathway. Our electrophysiological recordings demonstrate that the Ca2+ via an open TRPC4 channel activates PLCδ1 in the physiological range, and it causes the decrease of current amplitude. The existence of PLCδ1 accelerated PIP2 depletion when the channel was activated by an agonist. Interestingly, PLCδ1 mutants which have lost the ability to regulate PIP2 level failed to reduce the TRPC4 current amplitude. Our results demonstrate that TRPC4 self-regulates its activity by allowing Ca2+ ions into the cell and promoting the PIP2 hydrolyzing activity of PLCδ1.

BACKGROUND: Low-density lipoprotein cholesterol (LDL-C) is a major risk factor in atherogenesis and coronary heart disease as well as a primary target of lipid-lowering therapy. LDL-C concentration by direct homogenous assay was compared with that of the Friedewald formula, which is widely used in spite of its limitations. METHODS: Between February and March 2002, we analyzed total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and LDL-C levels in 1, 161 subjects (601 men and 560 women). They were classified according to cutpoints of the National Cholesterol Education Program Adult Treatment Panel III guidelines. The LDL-C results by direct method and the Friedewald formula were compared according to the TG levels and their medical decision values. RESULTS: Overall results of the direct method (Y) and the Friedewald formula (X) were highly correlated (Y=0.90X+13.62, r=0.9225). LDL-C by the Friedewald formula, however, showed a tendency of underestimation at higher TG levels. The results of the direct method were significantly different compared with those of the Friedewald formula when TG > or =200 mg/dL (P<0.05). Although the agreement between the two methods for LDL-C was within an acceptable range, it was relatively poor from near optimal to higher levels of LDL-C compared with optimal levels. CONCLUSIONS: The Friedewald formula is unsatisfactory for clinical purposes, because the levels of LDL-C are unreliable at the TG levels > or =200 mg/dL. Therefore, a direct determination method with better analytical performance is required. A fully automated homogenous assay seems to improve the determination of LDL-C, and may have a role in the diagnosis and management of hyperlipidemic patients.
Adult ; Atherosclerosis ; Cholesterol* ; Coronary Disease ; Diagnosis ; Education ; Humans ; Lipoproteins* ; Male ; Risk Factors ; Triglycerides

BACKGROUND: Early escharectomy has been shown to improve survival rates and treatment outcomes in major burn patients. However, its mechanism, especially in human immune systems, has not been fully elucidated. This observational study, focusing on cytokines, was conducted to assess changes in the levels of tumor necrosis factor alpha (TNF alpha) and interleukin-10 (IL-10) in major burn patients that underwent early tissue excision. METHODS: Seventeen ASA physical status II or III adults major burn patients, admitted to general surgery for burn wound care, were initially recruited. When early escharectomy was scheduled, a series of blood samples was obtained four times at 72 and 24 hours preop and at 24 and 72 hours postop. Changing levels of TNF alpha and IL-10 were measured by quantitative sandwich immnuoassay. RESULTS: Subjects suffered from 70% TBSA burns. Both cytokines demonstrated a significant tendency to increase in the blood during the study period. Although they temporarily decreased 24 hours after surgery, this effect did not last. CONCLUSIONS: Burn injury certainly increases cytokine response. Early escharectomy appears to decrease the pro and anti-inflammatory cytokines only temporarily. It did not seem to have any long term effect in the human immune system in major burn patients, probably due to the complex nature of the injury.
Adult ; Burns* ; Cytokines* ; Humans ; Immune System ; Interleukin-10 ; Observational Study ; Survival Rate ; Tumor Necrosis Factor-alpha ; Wounds and Injuries