The Transient Receptor Potential Protein Homologue TRPC4/5 as a Candidate for the Nonselective Cationic Channel Activated by Muscarinic Stimulation in the Murine Stomach

The transient receptor potential protein homologue TRPC5 was reported as a molecular identity for the muscarinic receptor-activated nonselective cationic channel (NSCC) in the murine stomach smooth muscle. The canonical, or classical, transient receptor potential proteins, TRPC4 and TRPC5, were suggested as members of the same subfamily of TRPC channels and to be coexpressed as a heteromultimer of both TRPC as well as a homotetramer of each TRPC protein. Thus, we investigated whether the TRPC4 channel is also responsible for the NSCC activated by acetylcholine (ACh) or carbachol (CCh) using electrophysiological techniques. The TRPC channels were expressed in HEK293 cells. When murine TRPC4 channels (mTRPC4) were expressed, the current–voltage relationship of mTRPC4 was also similar to that recorded in native murine gastric myocytes or mTRPC5-expressing HEK cells. With 0.2 mM GTPγS in the pipette solution, the currents in mTRPC4-expressing cells were activated transiently like those in NSCC in the murine stomach and the expressed mTRPC5. The currents recorded in mTRPC4-expressing cells were inhibited by 1 mM La³⁺ and 100 μM flufenamate. The currents recorded in mTRPC4-expressing cells depended on the extracellular calcium concentration. From the above results, we suggest that mTRPC4/5 might be candidates for the NSCC activated by ACh or CCh in the murine stomach.     

TRPC4 is an essential component of the nonselective cation channel activated by muscarinic stimulation in mouse visceral smooth muscle cells

Classical transient receptor potential channels (TRPCs) are thought to be candidates for the nonselective cation channels (NSCCs) involved in pacemaker activity and its neuromodulation in murine stomach smooth muscle. We aimed to determine the role of TRPC4 in the formation of NSCCs and in the generation of slow waves. At a holding potential of -60 mV, 50 mM carbachol (CCh) induced INSCC of amplitude [500.8+/-161.8 pA (n=8)] at -60 mV in mouse gastric smooth muscle cells. We investigated the effects of commercially available antibodies to TRPC4 on recombinant TRPC4 expressed in HEK cells and CCh-induced NSCCs in gastric smooth muscle cells. TRPC4 currents in HEK cells were reduced from 1525.6+/-414.4 pA (n=8) to 146.4+/-83.3 pA (n=10) by anti-TRPC4 antibody and INSCC amplitudes were reduced from 230.9+/-36.3 pA (n=15) to 49.8+/-11.8 pA (n=9). Furthermore, INSCC in the gastric smooth muscle cells of TRPC4 knockout mice was only 34.4+/-10.4 pA (n=8) at -60 mV. However, slow waves were still present in the knockout mice. Our data suggest that TRPC4 is an essential component of the NSCC activated by muscarinic stimulation in the murine stomach.     

Functional evidence for Na+-activated K+ channels in circular smooth muscle of the opossum lower esophageal sphincter

Na(+) reduction induces contraction of opossum lower esophageal sphincter (LES) circular smooth muscle strips in vitro; however, the mechanism(s) by which this occurs is unknown. The purpose of the present study was to investigate the electrophysiological effects of low Na(+) on opossum LES circular smooth muscle. In the presence of atropine, quanethidine, nifedipine, and substance P, conventional intracellular electrodes recorded a resting membrane potential (RMP) of -37.5 +/- 0.9 mV (n = 4). Decreasing [Na(+)] from 144.1 to 26.1 mM by substitution of equimolar NaCl with choline Cl depolarized the RMP by 7.1 +/- 1.1 mV. Whole cell patch-clamp recordings revealed outward K(+) currents that began to activate at -60 mV using 400-ms stepped test pulses (-120 to +100 mV) with increments of 20 mV from holding potential of -80 mV. Reduction of [Na(+)] in the bath solution inhibited K(+) currents in a concentration-dependent manner. Single channels with conductance of 49-60 pS were recorded using cell-attached patch-clamp configurations. The channel open probability was significantly decreased by substitution of bath Na(+) with equimolar choline. A 10-fold increase of [K(+)] in the pipette shifted the reversal potential of the single channels to the positive by -50 mV. These data suggest that Na(+)-activated K(+) channels exist in the circular smooth muscle of the opossum LES.     

TRPC4 and TRPC5: receptor-operated Ca2+-permeable nonselective cation channels

The seven mammalian channels from the classical (TRPC) subfamily of transient receptor potential (TRP) channels are thought to be receptor-operated cation channels activated in a phospholipase C (PLC)-dependent manner. Based on sequence similarity, TRPC channels can be divided into four subgroups. Group 4 comprises TRPC4 and TRPC5, and is most closely related to group 1 (TRPC1). The functional properties observed following heterologous expression of TRPC4 or TRPC5 in mammalian cells are contradictory and, therefore, controversial. In our hands, and in several independent studies, both channels, probably as homotetramers, form receptor-operated, Ca2+-permeable, nonselective cation channels activated independently of inositol 1,4,5-trisphosphate (InsP(3)) receptor activation or Ca2+ store-depletion. As heteromultimers with TRPC1, TRPC4 and TRPC5 form receptor-operated, Ca2+-permeable, nonselective cation channels with biophysical properties distinct from homomeric TRPC4 or TRPC5. In other studies, TRPC4 and TRPC5 have been shown to be store-operated channels, with moderate to high Ca2+ permeabilities. At present there is no clear explanation for these major differences in functional properties. To date, little is known as to which native cation channels are formed by TRPC4 and TRPC5. Endothelial cells from TRPC4(-/-) mice lack a highly Ca2+-permeable, store-dependent current, and data support a role for TRPC4 in endothelium-mediated vasorelaxation. A similar current in adrenal cortical cells is reduced by TRPC4 antisense. From similarities in the properties of the currents and expression of appropriate isoforms in the tissues, it is likely that heteromultimers of TRPC1 and TRPC4 or TRPC5 form receptor-operated nonselective cation channels in central neurones, and that TRPC4 contributes to nonselective cation channels in intestinal smooth muscle.     

Inhibition of diacylglycerol-sensitive TRPC channels by synthetic and natural steroids

TRPC channels are a family of nonselective cation channels that regulate ion homeostasis and intracellular Ca(2+) signaling in numerous cell types. Important physiological functions such as vasoregulation, neuronal growth, and pheromone recognition have been assigned to this class of ion channels. Despite their physiological relevance, few selective pharmacological tools are available to study TRPC channel function. We, therefore, screened a selection of pharmacologically active compounds for TRPC modulating activity. We found that the synthetic gestagen norgestimate inhibited diacylglycerol-sensitive TRPC3 and TRPC6 with IC(50)s of 3-5 μM, while half-maximal inhibition of TRPC5 required significantly higher compound concentrations (>10 μM). Norgestimate blocked TRPC-mediated vasopressin-induced cation currents in A7r5 smooth muscle cells and caused vasorelaxation of isolated rat aorta, indicating that norgestimate could be an interesting tool for the investigation of TRP channel function in native cells and tissues. The steroid hormone progesterone, which is structurally related to norgestimate, also inhibited TRPC channel activity with IC(50)s ranging from 6 to 18 μM but showed little subtype selectivity. Thus, TRPC channel inhibition by high gestational levels of progesterone may contribute to the physiological decrease of uterine contractility and immunosuppression during pregnancy.     

TRPC3 mediates pyrimidine receptor-induced depolarization of cerebral arteries

We tested the hypothesis that TRPC3, a member of the canonical transient receptor potential (TRP) family of channels, mediates agonist-induced depolarization of arterial smooth muscle cells (SMCs). In support of this hypothesis, we observed that suppression of arterial SMC TRPC3 expression with antisense oligodeoxynucleotides significantly decreased the depolarization and constriction of intact cerebral arteries in response to UTP. In contrast, depolarization and contraction of SMCs induced by increased intravascular pressure, i.e., myogenic responses, were not altered by TRPC3 suppression. Interestingly, UTP-evoked responses were not affected by suppression of a related TRP channel, TRPC6, which was previously found to be involved in myogenic depolarization and vasoconstriction. In patch-clamp experiments, UTP activated a whole cell current that was greatly reduced or absent in TRPC3 antisense-treated SMCs. These results indicate that TRPC3 mediates UTP-induced depolarization of arterial SMCs and that TRPC3 and TRPC6 may be differentially regulated by receptor activation and mechanical stimulation, respectively.     

TRPC5 as a candidate for the nonselective cation channel activated by muscarinic stimulation in murine stomach

We investigated which transient receptor potential (TRP) channel is responsible for the nonselective cation channel (NSCC) activated by carbachol (CCh) in murine stomach with RT-PCR and the electrophysiological method. All seven types of TRP mRNA were detected in murine stomach with RT-PCR. When each TRP channel was expressed, the current-voltage relationship of mTRP5 was most similar to that recorded in murine gastric myocytes. mTRP5 showed a conductance order of Cs(+) > K(+) > Na(+), similar to that in the murine stomach. With 0.2 mM GTPgammaS in the pipette solution, the current was activated transiently in both NSCC in the murine stomach and the expressed mTRP5. Both NSCC activated by CCh in murine stomach and mTRP5 were inhibited by intracellularly applied anti-G(q/11) antibody, PLC inhibitor U-73122, IICR inhibitor 2-aminoethoxydiphenylborate, and nonspecific cation channel blockers La(3+) and flufenamate. There were two other unique properties. Both the native NSCC and mTRP5 were activated by 1-oleoyl-2-acetyl-sn-glycerol. Without the activation of NSCC by CCh, the NSCC in murine stomach was constitutively active like mTRP5. From the above results, we suggest that mTRP5 might be a candidate for the NSCC activated by ACh or CCh in murine stomach.     

Increased vascular smooth muscle contractility in TRPC6-/- mice

Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6(-)(/)(-) smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6(-/-) smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.     

In vivo TRPC functions in the cardiopulmonary vasculature

Cardiovascular diseases are the leading cause of death in the industrialized countries. The cardiovascular system includes the systemic blood circulation, the heart and the pulmonary circulation providing sufficient blood flow and oxygen to peripheral tissues and organs according to their metabolic demand. This review focuses on three major cell types of the cardiovascular system: myocytes of the heart as well as smooth muscle cells and endothelial cells from the systemic and pulmonary circulation. Ion channels initiate and regulate contraction in all three cell types, and the identification of their genes has significantly improved our knowledge of signal transduction pathways in these cells. Among the ion channels expressed in smooth muscle cells, cation channels of the TRPC family allow for the entry of Na(+) and Ca(2+). Physiological functions of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 in the cardiovascular system, dissected by down-regulating channel activity in isolated tissues or by the analysis of gene-deficient mouse models, are reviewed. Possible functional roles and physiological regulation of TRPCs as homomeric or heteromeric channels in these cell types are discussed. Moreover, TRP channels may also be responsible for pathophysiological processes of the cardiovascular system like hypertension as well as cardiac hypertrophy and increased endothelial permeability.     

TRPC4 and TRPC5: receptor-operated Ca-permeable nonselective cation channels

The seven mammalian channels from the classical (TRPC) subfamily of transient receptor potential (TRP) channels are thought to be receptor-operated cation channels activated in a phospholipase C (PLC)-dependent manner. Based on sequence similarity, TRPC channels can be divided into four subgroups. Group 4 comprises TRPC4 and TRPC5, and is most closely related to group 1 (TRPC1). The functional properties observed following heterologous expression of TRPC4 or TRPC5 in mammalian cells are contradictory and, therefore, controversial. In our hands, and in several independent studies, both channels, probably as homotetramers, form receptor-operated, Ca²⁺-permeable, nonselective cation channels activated independently of inositol 1,4,5-trisphosphate (InsP₃) receptor activation or Ca²⁺ store-depletion. As heteromultimers with TRPC1, TRPC4 and TRPC5 form receptor-operated, Ca²⁺-permeable, nonselective cation channels with biophysical properties distinct from homomeric TRPC4 or TRPC5. In other studies, TRPC4 and TRPC5 have been shown to be store-operated channels, with moderate to high Ca²⁺ permeabilities. At present there is no clear explanation for these major differences in functional properties. To date, little is known as to which native cation channels are formed by TRPC4 and TRPC5. Endothelial cells from TRPC4^−/− mice lack a highly Ca²⁺-permeable, store-dependent current, and data support a role for TRPC4 in endothelium-mediated vasorelaxation. A similar current in adrenal cortical cells is reduced by TRPC4 antisense. From similarities in the properties of the currents and expression of appropriate isoforms in the tissues, it is likely that heteromultimers of TRPC1 and TRPC4 or TRPC5 form receptor-operated nonselective cation channels in central neurones, and that TRPC4 contributes to nonselective cation channels in intestinal smooth muscle.     

Molecular determinants of PKA-dependent inhibition of TRPC5 channel

Canonical transient receptor potential (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that are widely expressed in numerous cell types. Here, we demonstrate a new mechanism of TPRC isofom 5 (TRPC5) regulation, via cAMP signaling via Gα(s). Monovalent cation currents in human embryonic kidney-293 cells transfected with TRPC5 were induced by G protein activation with intracellular perfusion of GTPγS or by muscarinic stimulation. This current could be inhibited by a membrane-permeable analog of cAMP, 8-bromo-cAMP, by isoproterenol, by a constitutively active form of Gα(s) [Gα(s) (Q227L)], and by forskolin. These inhibitory effects were blocked by the protein kinase A (PKA) inhibitors, KT-5720 and H-89, as well as by two point mutations at consensus PKA phosphorylation sites on TRPC5 (S794A and S796A). Surface expression of several mutated versions of TRPC5, quantified using surface biotinylation, were not affected by Gα(s) (Q227L), suggesting that trafficking of this channel does not underlie the regulation we report. This mechanism of inhibition was also found to be important for the closely related channel, TRPC4, in particular for TRPC4α, although TRPC4β was also affected. However, this form of regulation was not found to be involved in TRPC6 and transient receptor potential vanilloid 6 function. In murine intestinal smooth muscle cells, muscarinic stimulation-induced cation currents were mediated by TRPC4 (>80%) and TRPC6. In murine intestinal smooth muscle cells, 8-bromo-cAMP, adrenaline, and isoproterenol decreased nonselective cation currents activated by muscarinic stimulation or GTPγS. Together, these results suggest that TRPC5 is directly phosphorylated by G(s)/cAMP/PKA at positions S794 and S796. This mechanism may be physiologically important in visceral tissues, where muscarinic receptor and β(2)-adrenergic receptor are involved in the relaxation and contraction of smooth muscles.     

Non-Selective Cation Channels Mediate Chloroquine-Induced Relaxation in Precontracted Mouse Airway Smooth Muscle

Bitter tastants can induce relaxation in precontracted airway smooth muscle by activating big-conductance potassium channels (BKs) or by inactivating voltage-dependent L-type Ca²⁺ channels (VDLCCs). In this study, a new pathway for bitter tastant-induced relaxation was defined and investigated. We found nifedipine-insensitive and bitter tastant chloroquine-sensitive relaxation in epithelium-denuded mouse tracheal rings (TRs) precontracted with acetylcholine (ACH). In the presence of nifedipine (10 µM), ACH induced cytosolic Ca²⁺ elevation and cell shortening in single airway smooth muscle cells (ASMCs), and these changes were inhibited by chloroquine. In TRs, ACH triggered a transient contraction under Ca²⁺-free conditions, and, following a restoration of Ca²⁺, a strong contraction occurred, which was inhibited by chloroquine. Moreover, the ACH-activated whole-cell and single channel currents of non-selective cation channels (NSCCs) were blocked by chloroquine. Pyrazole 3 (Pyr3), an inhibitor of transient receptor potential C3 (TRPC3) channels, partially inhibited ACH-induced contraction, intracellular Ca²⁺ elevation, and NSCC currents. These results demonstrate that NSCCs play a role in bitter tastant-induced relaxation in precontracted airway smooth muscle.     

Ca(2+) signaling by TRPC3 involves Na(+) entry and local coupling to the Na(+)/Ca(2+) exchanger

TRPC3 has been suggested as a key component of phospholipase C-dependent Ca(2+) signaling. Here we investigated the role of TRPC3-mediated Na(+) entry as a determinant of plasmalemmal Na(+)/Ca(2+) exchange. Ca(2+) signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na(+), in that carbachol-stimulated Ca(2+) entry into TRPC3 expressing cells was significantly suppressed when extracellular Na(+) was reduced to 5 mm. Moreover, KB-R9743 (5 microm) an inhibitor of the Na(+)/Ca(2+) exchanger (NCX) strongly suppressed TRPC3-mediated Ca(2+) entry but not TRPC3-mediated Na(+) currents. NCX1 immunoreactivity was detectable in HEK293 as well as in TRPC3-overexpressing HEK293 cells, and reduction of extracellular Na(+) after Na(+) loading with monensin resulted in significant rises in intracellular free Ca(2+) (Ca(2+)(i)) of HEK293 cells. Similar rises in Ca(2+)(i) were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na(+) subsequent to stimulation with carbachol. These increases in Ca(2+)(i) were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 microm), chelation of extracellular Ca(2+), or dominant negative suppression of TRPC3 channel function. This suggests that Ca(2+) entry into TRPC3-expressing cells involves reversed mode Na(+)/Ca(2+) exchange. Cell fractionation experiments demonstrated co-localization of TRPC3 and NCX1 in low density membrane fractions, and co-immunoprecipitation experiments provided evidence for association of TRPC3 and NCX1. Glutathione S-transferase pull-down experiments revealed that NCX1 interacts with the cytosolic C terminus of TRPC3. We suggest functional and physical interaction of nonselective TRPC cation channels with NCX proteins as a novel principle of TRPC-mediated Ca(2+) signaling.     

TRPC6 is a candidate channel involved in receptor-stimulated cation currents in A7r5 smooth muscle cells

To investigate thepossible role of members of the mammalian transient receptor potential(TRP) channel family (TRPC1-7) in vasoconstrictor-inducedCa²⁺ entry in vascular smooth muscle cells, we studied[Arg⁸]-vasopressin (AVP)-activated channels in A7r5aortic smooth muscle cells. AVP induced an increase in free cytosolicCa²⁺ concentration ([Ca²⁺]_i)consisting of Ca²⁺ release and Ca²⁺ influx.Whole cell recordings revealed the activation of a nonselective cationcurrent with a doubly rectifying current-voltage relation strikinglysimilar to those described for some heterologously expressed TRPCisoforms. The current was also stimulated by direct activation of Gproteins as well as by activation of the phospholipase C-coupledplatelet-derived growth factor receptor. Currents were not activated bystore depletion or increased [Ca²⁺]_i.Application of 1-oleoyl-2-acetyl-sn-glycerol stimulated the current independently of protein kinase C, a characteristic property ofthe TRPC3/6/7 subfamily. Like TRPC6-mediated currents, cation currentsin A7r5 cells were increased by flufenamate. Northern hybridizationrevealed mRNA coding for TRPC1 and TRPC6. We therefore suggest thatTRPC6 is a molecular component of receptor-stimulated Ca²⁺-permeable cation channels in A7r5 smooth muscle cells.

     

Molecular Linkage of TRP Proteins to Smooth Muscle Receptor-Operated Ca2+-Permeable Cationic Channels

The molecular mechanisms underlying Ca²⁺ entry evoked by cell surface receptors in smooth muscle have long been enigmatic, but an important breakthrough has been made by recent investigations on mammalian homologues of Drosophila transient receptor potential (TRP) protein. There is now growing evidence that TRPC6 plays an integrative role in vascular tone regulation, Ca²⁺ entry channels activated by the sympathetic nerve stimulation, vasoactive peptides, and mechanosensitive mechanisms. Other TRPC isoforms, such as TRPC1 and TRPC4 (and perhaps TRPC5), are also expressed abundantly in smooth muscle and may contribute to muscle contraction, cell proliferation, and cholinergic control of the gut motility. This paper briefly overviews the current knowledge about these TRP proteins in smooth muscle physiology.     

Activation of a TRPC3-dependent cation current through the neurotrophin BDNF

Nonvoltage-gated cation currents, which are activated following stimulation of phospholipase C (PLC), appear to be major modes for Ca2+ and Na+ entry in mammalian cells. The TRPC channels may mediate some of these conductances since their expression in vitro leads to PLC-dependent cation influx. We found that the TRPC3 protein was highly enriched in neurons of the central nervous system (CNS). The temporal and spatial distribution of TRPC3 paralleled that of the neurotrophin receptor TrkB. Activation of TrkB by brain-derived nerve growth factor (BDNF) led to production of a PLC-dependent, nonselective cation conductance in pontine neurons. Evidence is provided that TRPC3 contributes to this current in vivo. Thus, activation of TrkB and PLC leads to a TRPC3-dependent cation influx in CNS neurons.     

Molecular and electrophysiological characteristics of K+ conductance sensitive to acidic pH in aortic smooth muscle cells of WKY and SHR

Changes in K(+) conductances and their contribution to membrane depolarization in the setting of an acidic pH environment have been studied in myocytes from aortic smooth muscle cells of spontaneously hypertensive rats (SHR) compared with those from Wistar-Kyoto (WKY) rats. The resting membrane potential (RMP) of aortic smooth muscle at extracellular pH (pH(o)) of 7.4 was significantly more depolarized in SHR than in WKY rats. Acidification to pH(o) 6.5 made this difference in RMP between SHR and WKY rats more significant by further depolarizing the SHR myocytes. Large-conductance Ca(2+)-activated K(+) (BK) currents, which were markedly suppressed by acidification, were larger in aortic myocytes of SHR than in those of WKY rats. In contrast, acid-sensitive, non-BK currents were smaller in SHR. Western blot analyses showed that expression of BK-alpha- and -beta(1) subunits in SHR aortas was upregulated and comparable with those in WKY rats, respectively. Additional electrophysiological and molecular studies showed that pH- and halothane-sensitive two-pore domain weakly inward rectifying K(+) channel (TWIK)-like acid-sensitive K(+) (TASK) channel subtypes were functionally expressed in aortas, and TASK1 expression was significantly higher in WKY than in SHR. Although the background current through TASK channels at normal pH(o) (7.4) was small and may not contribute significantly to the regulation of RMP, TASK channel activation by halothane or alkalization (pH(o) 8.0) induced significant hyperpolarization in WKY but not in SHR. In conclusion, the larger depolarization and subsequent abnormal contractions after acidification in aortic myocytes in the setting of SHR hypertension are mainly attributable to the larger contribution of BK current to the total membrane conductance than in WKY aortas.     

Stretch-independent activation of the mechanosensitive cation channel in oocytes of Xenopus laevis

Oocytes of the South African clawed toad Xenopus laevis possess in their plasma membrane a so-called stretch-activated cation channel (SAC) which is activated by gently applying positive or negative pressure (stretch) to the membrane patch containing the channels. We show here that this mechanosensitive channel acted as a spontaneously opening, stretch-independent non-selective cation channel (NSCC) in more than half of the oocytes that we investigated. In 55% of cell-attached patches (total number of patches, 58) on 30 oocytes from several different donors, we found NSCC opening events. These currents were increased by elevating the membrane voltage or raising the temperature. NSCC and SAC currents shared some properties regarding the relative conductances of Na+>Li+>Ca2+, gating behaviour and amiloride sensitivity. Stretch-independent currents could be clearly distinguished from stretch induced SAC currents by their voltage and temperature dependence. Open events of NSCC increased strongly when temperature was raised from 21 to 27 degrees C. NSCC currents could be partly inhibited by high concentrations of extracellular Gd3+ and amiloride (100 and 500 microM, respectively). We further show exemplarily that NSCC can seriously hamper investigations when oocytes are used for the expression of foreign ion channels. In particular, NSCC complicated investigations on cation channels with small conductance as we demonstrate for a 4 pS epithelial Na+ channel (ENaC) from guinea pig distal colon. Our studies on NSCCs suggest the involvement of these channels in oocyte temperature response and ion transport regulation. From our results we suggest that NSCC and SAC currents are carried by one protein operating in different modes.