In most non-excitable cells, calcium (Ca(2+)) release from the inositol 1,4,5-trisphosphate (InsP(3))-sensitive intracellular Ca(2+) stores is coupled to Ca(2+) influx through the plasma membrane Ca(2+) channels whose molecular composition is poorly understood. Several members of mammalian TRP-related protein family have been implicated to both receptor- and store-operated Ca(2+) influx. Here we investigated the role of the native transient receptor potential 3 (TRPC3) homologue in mediating the store- and receptor-operated calcium entry in A431 cells. We show that suppression of TRPC3 protein levels by small interfering RNA (siRNA) leads to a significant reduction in store-operated calcium influx without affecting the receptor-operated calcium influx. With single-channel analysis, we further demonstrate that reduction of TRPC3 levels results in suppression of specific subtype of store-operated calcium channels and activation of store-independent channels. Our data suggest that TRPC3 is required for the formation of functional store-operated channels in A431 cells.
Activation of PLC-linked intracellular signaling cascades in the non-excitable cells evokes the release of calcium ions from the inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and Ca2+ entry in the cytosol via Ca2+-channels of plasma membrane. The properties and molecular identity of these channels are now under intense investigation. It is speculated that mammalian proteins belonging to the TRP-related family take part in either receptor- and store- dependent entry, though data linking the specific TRP proteins and any endogenous Ca 2+- channel are very scarce. Thus we aimed to study the role of TRPC3 in the formation of receptor- and store- operated calcium entry pathways in A431 cells. Both whole-cell current recordings and fluorescent measurements of intracellular Ca2+ concentration have shown that partial inhibition of TRPC3 expression with small interfering RNAs (siRNA) suppresses the store-dependent Ca2+ entry, but does not affect the receptor-mediated Ca2+ entry. The investigations on the single-channel level revealed that TRPC3 suppression leads to the disappearance of one of the types of store operated Ca2+- channels in the plasma membrane and appearance of a new type of a store-independent channel in it. It might indicate that TRPC3 protein is needed for the functioning of store-dependent channel in A431 cells.
UNLABELLED: The aim of this study was to determine the effects of desethyl-amiodarone (DEA), the major metabolite of the class III antiarrhythmic drug amiodarone, on human ether-à-go-go-related gene (hERG) encoded potassium channel current. MATERIALS AND METHODS: Whole-cell patch clamp recordings were made at 37 degrees C of ionic current (I(hERG)) carried by recombinant hERG channels expressed in HEK-293 cells. RESULTS: Desethyl-amiodarone inhibited I(hERG) with a half-maximal inhibitory concentration of approximately 158 nmol/L, compared with approximately 47 nmol/L for amiodarone. The inhibitory action of DEA on I(hERG) was contingent on channel gating, showing significant time and voltage dependence. Desethyl-amiodarone also produced an approximately -9 mV shift in the voltage dependence of activation of I(hERG); however, there was no significant preference for activated over inactivated channels. CONCLUSIONS: Because hERG underlies native cardiac "I(Kr)" channels, hERG/I(Kr) inhibition by DEA as well as amiodarone may contribute to the overall effects of amiodarone administration on cardiac repolarization.
Paroxysmal atrial fibrillation (PAF) is the most common cardiac arrhythmia, conveying a stroke risk comparable to persistent AF. It poses a significant diagnostic challenge given its intermittency and potential brevity, and absence of symptoms in most patients. This pilot study introduces a novel biomarker for early PAF detection, based upon analysis of sinus rhythm ECG waveform complexity. Sinus rhythm ECG recordings were made from 52 patients with ( n = 28) or without ( n = 24) a subsequent diagnosis of PAF. Subjects used a handheld ECG monitor to record 28-second periods, twice-daily for at least 3 weeks. Two independent ECG complexity indices were calculated using a Lempel-Ziv algorithm: R-wave interval variability (beat detection, BD) and complexity of the entire ECG waveform (threshold crossing, TC). TC, but not BD, complexity scores were significantly greater in PAF patients, but TC complexity alone did not identify satisfactorily individual PAF cases. However, a composite complexity score ( h -score) based on within-patient BD and TC variability scores was devised. The h -score allowed correct identification of PAF patients with 85% sensitivity and 83% specificity. This powerful but simple approach to identify PAF sufferers from analysis of brief periods of sinus-rhythm ECGs using hand-held monitors should enable easy and low-cost screening for PAF with the potential to reduce stroke occurrence.