Mammalian members of the classical transient receptor potential channel (TRPC) subfamily (TRPC1-7and Gd 3؉ have opposite effects on whole cell currents through TRPC5 and TRPC6 channels. The potentiation of TRPC4 and TRPC5 by micromolar La 3؉ at extracellular sites close to the pore mouth is a promising tool for identifying the involvement of these isoforms in receptor-operated cation conductances of native cells. Mammalian isoforms of the classical transient receptor potential channel (TRPC)1 subfamily, TRPC1-7, are likely candidates for cation channels mediating phospholipase C-dependent, receptor-operated Ca 2ϩ influx (for reviews, see Refs. 1-5). The properties and activation mechanisms of TRPCs have been studied extensively in heterologous expression systems. By contrast, relatively little information is available on their role in native cells. Two recent studies (6, 7) report that endogenous receptor-stimulated cation currents in vascular smooth muscle cells show properties identical to those described for heterologously expressed TRPC6. Therefore, precise knowledge of the properties of heterologously expressed TRPC channels may be essential to evaluate the involvement of TRPC proteins in receptor-operated cation conductances in native cells.Structurally, TRP channels, like many other cation channels, have been proposed to have six transmembrane segments (S1-S6), intracellular N and C termini, and a pore-forming reentrant loop between S5 and S6. Based on amino acid sequence similarity, the mammalian members of the TRPC subfamily can be subdivided into four groups (4, 8): TRPC1 (group 1), TRPC2 (group 2), TRPC3/6/7 (group 3), and TRPC4/5 (group 4). This subdivision is also supported by functional data. One of the major functional criteria is the mechanism of channel activation. From studies in heterologous expression systems, it is undisputed that receptor-mediated stimulation of phospholipase C is a key event in the activation of all TRPC isoforms. Evidence indicates that currents mediated by TRPC3, TRPC6, or TRPC7 can be activated by diacylglycerol (DAG) independently of protein kinase C (9 -12), although there is some controversy regarding the physiological significance of this stimulation (13). By contrast, TRPC4 and TRPC5 are not activated by DAG, and some evidence indicates that unidentified components of the phospholipase C pathway other than DAG or store depletion activate the channels (14 -16). Other evidence supports a store-dependent activation mechanism (17)(18)(19)(20). TRPC1 has been reported to be a store-dependent channel in some studies (21-23), but doubts have been raised as to whether TRPC1 expression results in the formation of functional plasma membrane channels in mammalian cells (16,24,25). The few data available support a store-operated activation mechanism for TRPC2 (26, 27), but, here again, other evidence suggests that TRPC2 does not form functional channels in all tissues (28). Data from our group, confirmed in several independent laboratories, indicate that several biophysical fe...

To investigate the possible role of members of the mammalian transient receptor potential (TRP) channel family (TRPC1-7) in vasoconstrictor-induced Ca(2+) entry in vascular smooth muscle cells, we studied [Arg(8)]-vasopressin (AVP)-activated channels in A7r5 aortic smooth muscle cells. AVP induced an increase in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) consisting of Ca(2+) release and Ca(2+) influx. Whole cell recordings revealed the activation of a nonselective cation current with a doubly rectifying current-voltage relation strikingly similar to those described for some heterologously expressed TRPC isoforms. The current was also stimulated by direct activation of G proteins as well as by activation of the phospholipase Cgamma-coupled platelet-derived growth factor receptor. Currents were not activated by store depletion or increased [Ca(2+)](i). Application of 1-oleoyl-2-acetyl-sn-glycerol stimulated the current independently of protein kinase C, a characteristic property of the TRPC3/6/7 subfamily. Like TRPC6-mediated currents, cation currents in A7r5 cells were increased by flufenamate. Northern hybridization revealed mRNA coding for TRPC1 and TRPC6. We therefore suggest that TRPC6 is a molecular component of receptor-stimulated Ca(2+)-permeable cation channels in A7r5 smooth muscle cells.

Large-conductance Ca(2+)-activated K(+) channels (BK channels) are highly expressed in human glioma cells. However, less is known about their biological function in these cells. We used the patch-clamp technique to investigate activation properties of BK channels and time-lapse microscopy to evaluate the role of BK channel activation in migration of 1321N1 human glioma cells. In whole cells, internal perfusion with a solution containing 500 nM free Ca(2+) and external application of the BK channel opener phloretin (100 micro M) shifted the activation threshold of BK channel currents toward more negative voltages of about -30 mV, which is close to the resting potential of the cells. The concentration of intracellular Ca(2+) in fura-2-loaded 1321N1 cells was measured to be 235+/-19 nM and was increased to 472+/-25 nM after treatment with phloretin. Phloretin and another BK channel opener NS1619 (100 micro M) reduced the migration velocity by about 50%. A similar reduction was observed following muscarinic stimulation of glioma cells with acetylcholine (100 micro M). The effects of phloretin, NS1619 and acetylcholine on cell migration were completely abolished by co-application of the specific BK channel blockers paxilline (5 micro M) and iberiotoxin (100 nM). The phloretin-induced increase in intracellular Ca(2+) was unaffected by the removal of extracellular Ca(2+) and co-application of paxilline. These findings indicate that glioma cell migration was inhibited through BK channel activation, independent of intracellular Ca(2+).

We have investigated the effects of histamine on the intracellular calcium concentration ([Ca2+]i) of cultured rat cerebellar astrocytes using fura‐2‐based Ca2+ imaging microscopy. Most of the cells responded to the application of histamine with an increase in [Ca2+]i which was antagonized by the H1 receptor blocker mepyramine. When histamine was applied for several minutes, the majority of the cells displayed a biphasic Ca2+ response consisting of an initial transient peak and a sustained component. In contrast to the initial transient [Ca2+]i response, the sustained, receptor‐activated increase in [Ca2+]i was rapidly abolished by chelation of extracellular Ca2+ or addition of Ni2+, Mn2+, Co2+ and Zn2+, but was unaffected by nifedipine, an antagonist of L‐type voltage‐activated Ca2+ channels. These data indicate that the sustained increase in [Ca2+]i was dependent on Ca2+ influx. When intracellular Ca2+ stores were emptied by prolonged application of histamine in Ca2+‐free conditions, Ca2+ re‐addition after removal of the agonist did not lead to an ‘overshoot’ of [Ca2+]i indicative of store‐operated Ca2+ influx. However, Ca2+ stores were refilled despite the absence of any substantial change in the fura‐2 signal. Depletion of intracellular Ca2+ stores using cyclopiazonic acid in Ca2+‐free saline and subsequent re‐addition of Ca2+ to the saline resulted in an increase in [Ca2+]i that was significantly enhanced in the presence of histamine. The results suggest that besides capacitative mechanisms, a non‐capacitative, voltage‐independent pathway is involved in histamine‐induced Ca2+ entry into cultured rat cerebellar astrocytes.

No abstract