Mammalian transient receptor potential channels (TRPCs) form a family of Ca2؉ -permeable cation channels currently consisting of seven members, TRPC1-TRPC7. These channels have been proposed to be molecular correlates for capacitative Ca 2؉ entry channels. There are only a few studies on the regulation and properties of the subfamily consisting of TRPC4 and TRPC5, and there are contradictory reports concerning the possible role of intracellular Ca 2؉ store depletion in channel activation. We therefore investigated the regulatory and biophysical properties of murine TRPC4 and TRPC5 (mTRPC4/5) heterologously expressed in human embryonic kidney cells. Activation of G q/11 -coupled receptors or receptor tyrosine kinases induced Mn 2؉ entry in fura-2-loaded mTRPC4/5-expressing cells. Accordingly, in whole-cell recordings, stimulation of G q/11 -coupled receptors evoked large, nonselective cation currents, an effect mimicked by infusion of guanosine 5-3-O-(thio)triphosphate (GTP␥S). However, depletion of intracellular Ca2؉ stores failed to activate mTRPC4/5. In inside-out patches, single channels with conductances of 42 and 66 picosiemens at ؊60 mV for mTRPC4 and mTRPC5, respectively, were stimulated by GTP␥S in a membrane-confined manner. Thus, mTRPC4 and mTRPC5 form nonselective cation channels that integrate signaling pathways from G-protein-coupled receptors and receptor tyrosine kinases independently of store depletion. Furthermore, the biophysical properties of mTRPC4/5 are inconsistent with those of I CRAC , the most extensively characterized store-operated current.

SUMMARY1. Ca2+ currents were recorded using the whole-cell mode of the patch-clamp technique from mouse pancreatic B-cells kept in culture for 1-4 days. B-cells were identified in the cell-attached mode by their response to a change in the glucose concentration from 3 to 15 or 20 mm or by their inward currents.2. Only one component of Ca2+ current was observed in these cells, which activated at potentials > -50 mV and was blocked by nitrendipine (5/M), and increased in amplitude by CGP 28392 (5 /tM).3. During maintained depolarizations the Ca2+ current inactivated considerably but not completely. Inactivation was most marked at potentials where the Ca2+ currents were large, but in general was slower for currents at potentials > 0 mV than at more negative potentials.4. Two-pulse experiments showed that the inactivation curve for the Ca2+ current was U-shaped, returning to unity at potentials approaching the Ca2+ equilibrium potential. Measurements of Ca2+ entry showed that inactivation was dependent on the amount of Ca2+ entering during the pre-pulse, independent of the pre-pulse potential.5. Ca2+ currents were not appreciably slowed when BAPTA, a faster buffer of Ca2+, replaced EGTA in the pipette solution.6. Replacement of Ca2+ in the external solution by Ba2+ increased the amplitude of the inward current and largely abolished inactivation. Large inward currents through Ca2+ channels were observed in the absence of divalent cations in the external solution (+ EGTA), which were presumably carried by Na+. These currents did not inactivate during 150 ms depolarizations, but were increased in amplitude by CGP 28392 (5 /M) and blocked by D600 (30 FM).7. The observations suggest that normal mouse pancreatic B-cells have only one type of Ca2+ channel which is dihydropyridine sensitive and inactivates by a mechanism which is almost purely Ca2+ dependent. Inactivation of the Ca2+ current will probably be important in the control of Ca2+ entry during glucose-induced electrical activity.

SUMMARY1. The effects of intracellular injection of Ca, EGTA and EGTA/Ca buffers on inward currents flowing through the Ca channel in Helix aspersa neurones were studied under voltage clamp.2. Inward currents were reversibly reduced by Ca injection. The extent of the reduction was dependent on the size and duration of the injection. Recovery from injection was exponential with a time constant around 18 s at 18-20 'C.3. In our salines, which contained tetraethylammonium chloride and 4-aminopyridine, no outward current was activated by Ca injection at the holding potential. A given Ca injection reduced the inward current by the same fraction in 25 mm-and 2-5 mM-Sr and also at different test potentials. We conclude that Ca injection does not activate an outward current. Mean resting ionized internal

We have identified prognostic factors and report progression to liver failure in 53% of NCIPH patients followed-up at our center. Our data supports a role for intestinal disease in the pathogenesis of intrahepatic portal vein occlusion leading to NCIPH.