Ionic channels formed by TRPC4.

TRPC4 (transient receptor potential canonical 4) is a member of the TRPC sub-family and, within this sub-family, TRPC4 is most closely related to TRPC5. A number of splice variants of TRPC4 have been identified, whereby TRPC4alpha and TRPC4beta appear to be the most abundant isoforms in various species. TRPC4alpha comprises six transmembrane segments and the N- and C-termini are located intracellularly. Additionally, TRPC4alpha shares other structural features with members of the TRPC sub-group, including ankyrin-like repeats, coiled-coil regions and binding sites for calmodulin and IP3 receptors. Three calmodulin-binding domains have been identified in the C-terminus of TRPC4alpha. TRPC4beta lack 84 amino acids in the C-terminus, which correspond to the last two calmodulin-binding sites of TRPCalpha. The first and last calmodulin-binding domains of TRPC4alpha overlap with binding sites for the N- and C-termini of IP3 receptors. The ionic channels formed by TRPC4 appear to be Ca(2+)-permeable, although there is a considerably discrepancy in the degree of Ca2+ selectivity. Studies with mice lacking TRPC4 (TRPC4(-/-)) suggest an important role for TRPC4 in supporting Ca2+ entry. The defect in Ca2+ entry in TRPC4(-/-) mice appears to be associated with a reduction of the vasorelaxation of arteries, vascular permeability in the lung and neurotransmitter release from thalamic dendrites.

Expression of CaT-like, a novel calcium-selective channel, correlates with the malignancy of prostate cancer.

The regulation of intracellular Ca(2+) plays a key role in the development and growth of cells. Here we report the cloning and functional expression of a highly calcium-selective channel localized on the human chromosome 7. The sequence of the new channel is structurally related to the gene product of the CaT1 protein cloned from rat duodenum and is therefore called CaT-like (CaT-L). CaT-L is expressed in locally advanced prostate cancer, metastatic and androgen-insensitive prostatic lesions but is undetectable in healthy prostate tissue and benign prostatic hyperplasia. Additionally, CaT-L is expressed in normal placenta, exocrine pancreas, and salivary glands. New markers with well defined biological function that correlate with aberrant cell growth are needed for the molecular staging of cancer and to predict the clinical outcome. The human CaT-L channel represents a marker for prostate cancer progression and may serve as a target for therapeutic strategies.

Rac and phosphatidylinositol 3-kinase regulate the protein kinase B in Fc epsilon RI signaling in RBL 2H3 mast cells.

FcepsilonRI signaling in rat basophilic leukemia cells depends on phosphatidylinositol 3-kinase (PI3-kinase) and the small GTPase Rac. Here, we studied the functional relationship among PI3-kinase, its effector protein kinase B (PKB), and Rac using inhibitors of PI3-kinase and toxins inhibiting Rac. Wortmannin, an inhibitor of PI3-kinase, blocked FcepsilonRI-mediated tyrosine phosphorylation of phospholipase Cgamma, inositol phosphate formation, calcium mobilization, and secretion of hexosaminidase. Similarly, Clostridium difficile toxin B, which inactivates all Rho GTPases including Rho, Rac and Cdc42, and Clostridium sordellii lethal toxin, which inhibits Rac (possibly Cdc42) but not Rho, blocked these responses. Stimulation of the FcepsilonRI receptor induced a rapid increase in the GTP-bound form of Rac. Whereas toxin B inhibited the Rac activation, PI3-kinase inhibitors (wortmannin and LY294002) had no effect on activation of Rac. In line with this, wortmannin had no effect on tyrosine phosphorylation of the guanine nucleotide exchange factor Vav. Wortmannin, toxin B, and lethal toxin inhibited phosphorylation of PKB on Ser(473). Similarly, translocation of the pleckstrin homology domain of PKB tagged with the green fluorescent protein to the membrane, which was induced by activation of the FcepsilonRI receptor, was blocked by inhibitors of PI3-kinase and Rac inactivation. Our results indicate that in rat basophilic leukemia cells Rac and PI3-kinase regulate PKB and suggest that Rac is functionally located upstream and/or parallel of PI3-kinase/PKB in FcepsilonRI signaling.

Single-channel currents through transient-receptor-potential-like (TRPL) channels.

Single-channel current recordings were used to examine the properties and modulation of Drosophila transient-receptor-potential-like (TRPL) channels transiently expressed in HEK and COS cells. Recombinant TRPL channels were constitutively active and characterized by a conductance of 104 pS in on-cell membrane patches with 115 mM Na+ and 2 mM Mg2+ in the pipette solution. In inside-out membrane patches exposed to 115 mM Na+ plus 2 mM Mg2+, 115 mM Na+ plus 10 mM Mg2+, 90 mM Ca2+ and 90 mM Ba2+ on both sides, the single-channel conductances were 72 pS, 36 pS, 48 pS and 46 pS, respectively. The single TRPL channel currents reversed close to 0 mV and displayed a linear voltage dependence between -120 mV and +120 mV. Removal of cations from the pipette and bath solutions abolished inward and outward currents, respectively. Similar currents were not observed in mock-transfected and native cells. The opening probability of TRPL channels increased by depolarizing the membrane and accounted for the outward rectification of whole-cell TRPL currents. In on-cell membrane patches, the TRPL channel activity was enhanced by cell dialysis of 300 microM guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) and by a rise of intracellular Ca2+ (>2 microM). Constitutively active TRPL channels depolarized the host cells to -10 mV and the membrane potential was restored by cell dialysis with 10 mM BAPTA. The present results suggest that TRPL forms non-selective cationic channels modulated by intracellular Ca2+ in mammalian cells.

Modulation of recombinant transient-receptor-potential-like (TRPL) channels by cytosolic Ca2+.

Whole-cell current recordings were used to examine the involvement of intracellular Ca2+ in the modulation of recombinant transient-receptor-potential like (TRPL) channels of Drosophila photoreceptor cells. TRPL was stably transfected in Chinese hamster ovary (CHO) cells and the expression of a calmodulin-binding protein with a molecular mass that corresponded to TRPL was demonstrated using calmodulin overlays. In cells expressing TRPL, ionic currents that were prominently outwardly rectifying were detected prior to activation of intracellular signalling pathways. The outwardly rectifying currents reversed close to 0 mV and did not occur after removal of permeant cations from the intracellular space. This suggests that TRPL forms non-selective cationic channels that appear to be constitutively active in mammalian cell lines. The TRPL channel currents were enhanced by manoeuvres that activate the phospholipase C (PLC) signalling pathway. These included activation of membrane receptors by thrombin, activation of G proteins by cell dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) and release of Ca2+ from intracellular stores by dialysis with inositol 1,4,5-trisphosphate (IP3). After complete depletion of Ca2+ stores, IP3 had no effect on TRPL currents, suggesting that IP3 does not activate recombinant TRPL channels directly. However, thapsigargin, which induces a rise of cytosolic Ca2+, increased TRPL channel currents. Cell dialysis with solutions containing various concentrations of Ca2+ enhanced TRPL currents in a dose-dependent manner (EC50=450 nM Ca2+). Conversely, chelation of cytosolic Ca2+ abolished TRPL channel currents. The present results indicate that the activity of recombinant TRPL channels expressed in mammalian cell lines is up-regulated by a rise of cytosolic Ca2+.

TRP4 (CCE1) protein is part of native calcium release-activated Ca2+-like channels in adrenal cells.

Mammalian TRP proteins have been implicated to function as ion channel subunits responsible for agonist-induced Ca(2+) entry. To date, TRP proteins have been extensively studied by heterologous expression giving rise to diverse channel properties and activation mechanisms including store-operated mechanisms. However, the molecular structure and the functional properties of native TRP channels still remain elusive. Here we analyze the properties of TRP4 (CCE1) channels in their native environment and characterize TRP expression patterns and store-operated calcium currents that are endogenous to bovine adrenal cells. We show by Northern blot analysis, immunoblots, and immunohistochemistry that TRP4 transcripts and TRP4 protein are present in the adrenal cortex but absent in the medulla. Correspondingly, bovine adrenal cortex cells express TRP4 abundantly. The only other TRP transcript found at considerable levels was TRP1, whereas TRP2, TRP3, TRP5(CCE2), and TRP6 were not detectable. Depletion of calcium stores with inositol 1,4,5-trisphosphate or thapsigargin activates store-operated ion channels in adrenal cells. These channels closely resemble calcium release-activated Ca(2+) (CRAC) channels. Expression of trp4(CCE1) cDNA in antisense orientation significantly reduces both, the endogenous CRAC-like currents and the amount of native TRP4 protein. These results demonstrate that TRP4 contributes essentially to the formation of native CRAC-like channels in adrenal cells.

Inhibition of calcium release-activated calcium current by Rac/Cdc42-inactivating clostridial cytotoxins in RBL cells.

Using large clostridial cytotoxins as tools, the role of Rho GTPases in activation of RBL 2H3 hm1 cells was studied. Clostridium difficile toxin B, which glucosylates Rho, Rac, and Cdc42 and Clostridium sordellii lethal toxin, which glucosylates Rac and Cdc42 but not Rho, inhibited the release of hexosaminidase from RBL cells mediated by the high affinity antigen receptor (FcepsilonRI). Additionally, toxin B and lethal toxin inhibited the intracellular Ca(2+) mobilization induced by FcepsilonRI-stimulation and thapsigargin, mainly by reducing the influx of extracellular Ca(2+). In patch clamp recordings, toxin B and lethal toxin inhibited the calcium release-activated calcium current by about 45%. Calcium release-activated calcium current, the receptor-stimulated Ca(2+) influx, and secretion were inhibited neither by the Rho-ADP-ribosylating C3-fusion toxin C2IN-C3 nor by the actin-ADP-ribosylating Clostridium botulinum C2 toxin. The data indicate that Rac and Cdc42 but not Rho are not only involved in late exocytosis events but are also involved in Ca(2+) mobilization most likely by regulating the Ca(2+) influx through calcium release-activated calcium channels activated via FcepsilonRI receptor in RBL cells.

Phenotype of a recombinant store-operated channel: highly selective permeation of Ca2+.

1. Genes related to trp (transient receptor potential) are proposed to encode store-operated channels. We examined the ionic permeation of recombinant channels formed by stable and transient expression of the TRP homologue bCCE1 in Chinese hamster ovary (CHO) cells (CHO(CCE1)) and rat basophilic leukaemia (RBL) cells, respectively. 2. Store-operated currents were activated in CHO(CCE1) cells by internal dialysis of IP3 under strong buffering of intracellular Ca2+. The action of IP3 was mimicked by thapsigargin but not by IP4. 3. With extracellular Ca2+, Na+ and Mg2+, the store-operated currents of CHO(CCE1) rectified inwardly in the presence of internal Cs+. Outward currents were not detected below +80 mV. Identical currents were recorded with external Ba2+ and also with no external Na+ and Mg2+. In the absence of external Mg2+, the inward currents showed an anomalous mole fraction behaviour between Ca2+ and Na+. Half-maximal inhibition of Na+ currents was observed with approximately 100 nM and full block with 2-5 microM external Ca2+. 4. In the parental CHO(-) cells, IP3 dialysis evoked inward currents that also displayed anomalous mole fraction behaviour between Ca2+ and Na+. However, half-maximal block of Na+ currents required 5 times higher Ca2+ concentrations in CHO(-) cells. Additionally, the density of Ca2+ and Na+ currents at -80 mV was 5 and 2 times larger in CHO(CCE1) cells, respectively. 5. In RBL cells, dialysis of IP3 evoked store-operated currents that showed 1.4-fold larger densities at -80 mV in cells expressing bCCE1. 6. The enhanced density of store-operated currents in CHO(CCE1) cells and in bCCE1-transfected RBL cells probably reflects the phenotype of CCE1. These results suggest a highly selective permeation of Ca2+ through recombinant channels formed by CCE1 either alone or in combination with endogenous channel proteins.

Ca2+-dependent interaction of the trpl cation channel and calmodulin.

The transient receptor potential-like ion channel from Drosophila melanogaster was originally identified as a calmodulin binding protein (Philips et al., 1992) involved in the dipterian phototransduction process. We used a series of fusion proteins and an epitope expression library of transient receptor potential-like fusion proteins to characterize calmodulin binding regions in the transient receptor potential-like channel through the use of [125I]calmodulin and biotinylated calmodulin and identified two distinct sites at the C-terminus of the transient receptor potential-like ion channel. Calmodulin binding site 1, predicted from searching of the primary structure for amphiphilic helices (Philips et al., 1992), covers a 16 amino acid sequence (S710-I725) and could only be detected through biotinylated calmodulin. Calmodulin binding site 2 comprises at least 13 amino acids (K859ETAKERFQRVAR871) and binds both [125I]calmodulin and biotinylated calmodulin. Both sites (i) bind calmodulin at least in a one to one stoichiometry, (ii) differ in their affinity for calmodulin revealing apparent Ki values of 12.3 nM (calmodulin binding site 1) and 1.7 nM (calmodulin binding site 2), respectively, (iii) bind calmodulin only in the presence of Ca2+ with 50% of site 1 and site 2, respectively, occupied by calmodulin in the presence of 0.1 microM (calmodulin binding site 1) and 3.3 microM Ca2+ (calmodulin binding site 2) and give evidence that (iv) a Ca2+-calmodulin-dependent mechanism contributes to transient receptor potential-like cation channel modulation when expressed in CHO cells.

A novel capacitative calcium entry channel expressed in excitable cells.

In addition to voltage-gated calcium influx, capacitative calcium entry (CCE) represents a major pathway for calcium entry into the cell. Here we report the structure, expression and functional properties of a novel CCE channel, TRP5. This channel is a member of a new subfamily of mammalian homologues of the Drosophila transient receptor potential (TRP) protein, now comprising TRP5 (also CCE2) and the structurally related CCE1 (also TRP4). Like TRP4, TRP5 forms ion channels mainly permeable for Ca2+ which are not active under resting conditions but can be activated by manoeuvres known to deplete intracellular calcium stores. Accordingly, dialysis of TRP5-expressing cells with inositol-(1,4,5)-trisphosphate evokes inward rectifying currents which reversed polarity at potentials more positive than +30 mV. Ca2+ store depletion with thapsigargin induced TRP5-mediated calcium entry dependent on the concentration of extracellular calcium, as seen by dual wavelength fura-2 fluorescence ratio measurements. TRP5 transcripts are expressed almost exclusively in brain, where they are present in mitral cells of the olfactory bulb, in lateral cerebellar nuclei and, together with TRP4 transcripts, in CA1 pyramidal neurons of the hippocampus, indicating the presence of CCE channels in excitable cells and their participation in neuronal calcium homeostasis.

Major histocompatibility complex (MHC) class I gene expression in single neurons of the central nervous system: differential regulation by interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha.

This study examined the effect of the pro-inflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on the induction of MHC class I-related genes in functionally mature brain neurons derived from cultures of dissociated rat hippocampal tissue. Patch clamp electrophysiology combined with single cell RT-PCR demonstrated that approximately 50% of the untreated neurons contained mRNA for MHC class I heavy chains, while, with few exceptions, the cells failed to transcribe beta2-microglobulin and TAP1/TAP2 gene transcripts. No constitutive expression of MHC class I protein was detectable by confocal laser microscopy on the surface of neurons. All neurons transcribed the alpha-chain of the interferon-type II receptor (binding IFN-gamma) along with the p55 receptor for TNF-alpha. Sustained exposure to IFN-gamma resulted in transcription of beta2-microglobulin and TAP1/TAP2 genes and MHC class I surface expression in a minor part of the neurons, but did not alter their electrophysiological activities as assessed by whole cell electrophysiology. Suppression of neuronal electric activity by the sodium channel blocker tetrodotoxin drastically increased to almost 100% IFN-gamma-mediated induction of MHC class I chains, of both TAP transporters, and of membrane expression of MHC class I protein. The effect of tetrodotoxin is at least partly reverted by the neurotransmitter glutamate. In contrast to IFN-gamma, treatment with TNF-alpha did neither upregulate TAP1/TAP2 nor beta2-microglobulin gene expression, but induced MHC class I heavy chain gene transcription in all neurons. Consequently, no MHC class I molecules were detectable on the membranes of TNF-alpha-treated neurons.

A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL.

Intracellular Ca2+ signalling evoked by Ca2+ mobilizing agonists, like angiotensin II in the adrenal gland, involves the activation of inositol(1,4,5)trisphosphate(InsP3)-mediated Ca2+ release from internal stores followed by activation of a Ca2+ influx termed capacitative calcium entry. Here we report the amino acid sequence of a functional capacitative Ca2+ entry (CCE) channel that supports inward Ca2+ currents in the range of the cell resting potential. The expressed CCE channel opens upon depletion of Ca2+ stores by InsP3 or thapsigargin, suggesting that the newly identified channel supports the CCE coupled to InsP3 signalling.

Induction of MHC class I genes in neurons.

Whether neurons express major histocompatibility complex (MHC) class I genes has not been firmly established. The techniques of confocal laser microscopy, patch clamp electrophysiology, and reverse transcriptase-polymerase chain reaction were combined here to directly examine the inducibility of MHC class I genes in individual cultured rat hippocampal neurons. Transcription of MHC class I genes was very rare in neurons with spontaneous action potentials. In electrically silent neurons, transcription was noted, with expression of beta 2-microglobulin under tighter control than in class I heavy chain molecules. Surface expression of class I molecules occurred only in electrically silent neurons treated with interferon gamma. Immunosurveillance by cytotoxic T cells may be focused on functionally impaired neurons.

Constitutive upregulation of calcium channel currents in rat phaeochromocytoma cells: role of c-fos and c-jun.

1. Northern blot analysis and cell transfection were used in conjunction with whole-cell current recordings to examine the involvement of the immediate early genes, c-fos and c-jun, in the expression of calcium channel currents. 2. Phaeochromocytoma cells (PC12 clone) were exposed to nerve growth factor (NGF) and to depolarizing concentrations of KCl for 60 min every day. Cells challenged with NGF developed extensive networks of neurites within 3 days. Cells depolarized periodically retained their undifferentiated morphology even after 5 days of treatment. 3. The maximal amplitude of high-voltage-activated calcium currents (ICa) increased from the control level of 117.8 +/- 48.3 (mean +/- S.D.) to 387.2 +/- 90.1 pA within 3 days of NGF treatment. omega-Conotoxin (5-10 microM) inhibited 24.6 +/- 8.5% of ICa in undifferentiated cells and 57.8 +/- 6.9% in NGF-treated cells. 4. The levels of c-fos and c-jun mRNAs increased transiently during each daily exposure to NGF. The level of c-fos mRNA also increased transiently during repeated KCl-induced depolarizations but c-jun mRNA remained low or absent. 5. Naive PC12 cells were transiently co-transfected with expression plasmids that contained the full length of c-fos and c-jun cDNA. After 2 days following transfection, the PC12 cells could be grouped according to the size of ICa. In 56% of cells, ICa was similar to control currents (106.1 +/- 37.4 pA). In the remaining 44% of cells, ICa showed a 2.2-fold enhancement with respect to control cells. Transfection of only c-fos had no effect on ICa but, in 24% of cells transfected with c-jun, ICa was 176.6 +/- 124.6 pA. Since periodic membrane depolarization induced c-fos but not c-jun mRNA, c-jun transfection was combined with a high-K+ treatment over 3 days. In 18% of treated cells, ICa was 3.7 times larger than control currents. Morphological differentiation was not observed in transfected cells. 6. In PC12 cells co-transfected with c-fos and c-jun or treated with high K+ after transfection of c-jun, omega-conotoxin (5-10 microM) inhibited 68.7 +/- 11.9% of ICa when the current amplitude was in the range of 200-600 pA. since similar concentrations of omega-conotoxin blocked 19.2 +/- 5.4% of ICa in control cells, the current increase induced by c-fos and c-jun was supported by up to 11-fold enhancement of the omega-conotoxin-sensitive component of ICa.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhancement of voltage-gated Ca2+ currents induced by daily stimulation of hippocampal neurons with glutamate.

The regulation of calcium channel currents (ICa) induced by daily stimulation (1 hr) with 10 microM glutamate was studied in full differentiated hippocampal cells in culture. We report a specific enhancement of the high-voltage-activated current type (HVA ICa) ongoing over days. The density of HVA ICa increased about twofold after the second glutamate session, and this enhancement was still observed after the fifth day of treatment, while low-voltage-activated calcium currents (LVA ICa) remained unchanged. During glutamate application, a transient increase of intracellular calcium (Cai) was observed, followed by a slow decay within 2-3 min, and substantial recovery in about 10 min. Similarly, Cai transients induced by periodic membrane depolarization mimicked the long-term effect of glutamate on ICa. These results demonstrate for the first time an increase of ICa in a time frame of days. Since the effect of glutamate on ICa was prevented by cycloheximide, neosynthesis of channel proteins presumably supports this enhancement.

Stable co-expression of calcium channel alpha 1, beta and alpha 2/delta subunits in a somatic cell line.

1. The high-voltage-activated L-type calcium channel is a multi-protein complex of alpha 1, alpha 2/delta, beta and gamma subunits. The alpha 1 subunit contains the voltage-dependent calcium-conducting pore. Chinese hamster ovary (CHO) cells were stably transfected with the complementary DNA of the alpha 1, beta and alpha 2/delta subunits. These subunits were not detected in wild-type CHO cells. 2. The alpha 1 (CaCh2b) subunit itself directed the expression of functional calcium channels which bound calcium channel blockers and showed voltage-dependent activation and inactivation. 3. The co-expression of the alpha 1 subunit with the beta subunit (CaB1 gene) enhanced the density of the dihydropyridine binding sites 2- to 3-fold and increased dihydropyridine-sensitive barium inward currents (IBa) up to 3.5-fold from -13.3 microA/cm2 (alpha 1 subunit) to -46.7 microA/cm2 (alpha 1 and beta subunits). 4. Co-expression of the beta subunit did not change the sensitivity of IBa towards dihydropyridines, but accelerated current activation and inactivation and shifted the half-maximal steady-state activation and inactivation to slightly more hyperpolarizing potentials. 5. The co-expression of the alpha 2/delta subunit together with alpha 1 and beta subunits accelerated the inactivation kinetics of the channel without a major effect on the other parameters. 6. These results indicate that the beta and alpha 2/delta subunit interact with the alpha 1 subunit and modulate thereby the properties of the alpha 1 subunit-dependent inward current.

Role of the GTP-binding protein Gs in the beta-adrenergic modulation of cardiac Ca channels.

In the heart, the guanosine 5'-triphosphate (GTP)-binding protein Gs is activated by hormone binding to beta-adrenergic receptors and stimulates the intracellular cyclic adenosine 3',5'-monophosphate (cAMP) pathway that leads to phosphorylation of L-type Ca channels by the cAMP-dependent protein kinase A. Additionally, Gs can modulate cardiac Ca channels directly in cell-free systems. In order to examine the question of whether these pathways could be separated functionally and whether they act independently or synergistically on L-type Ca channels in intact cells, the whole-cell Ca current (ICa) and the respective current density were measured in guinea-pig ventricular myocytes at 0 mV. The following results were obtained. First, typically, the ICa density increased from 12 to 40 microA/cm2 following application of 1 microM isoproterenol (ISP) to myocytes bathed in solutions containing 1.8 mM CaCl2. However, 1 microM ISP enhanced ICa only from 9 to 17 microA/cm2 after inhibition of the protein kinase A by dialysis of 0.5 mM Rp-cAMPs (the Rp-isomer of adenosine 3',5'-monophosphorothioate) in the presence of 0.5 mM GTP. Withdrawal of GTP from the dialysate attenuated the effects of ISP on ICa. Thus, Rp-cAMPS unmasks a GTP-dependent component of the beta-adrenergic stimulation of ICa, which probably reflects the direct stimulation of Ca channels by Gs under block of cAMP-dependent phosphorylation. Second, in cells under dialysis with 100 or 200 microM cAMP, bath application of 20-40 microM 3-isobutyl-1-methylxanthine (IBMX) enhanced the ICa density to about 41 microA/cm2 indicating saturation of the cAMP pathway. Under this condition, 1 microM ISP was without significant effect on ICa. This result may suggest that direct Gs stimulation is rather ineffective on Ca channels after maximal cAMP-dependent phosphorylation. Alternatively, maximal stimulation of the cAMP pathway may also interfere with the activation of the Gs pathway in intact myocytes. Third, simultaneous application of 1 microM ISP and 40 microM IBMX enhanced ICa up to densities of around 75 microA/cm2 during cell dialysis with 100 microM cAMP, an effect much stronger than that exerted by IBMX alone under similar conditions. Since it seems likely that Gs is activated more quickly, than the cAMP pathway during application of the ISP/IBMX mixture, the latter result suggests that a direct effect of Gs may act to prime L-type Ca channels for cAMP-dependent phosphorylation during beta-adrenergic stimulation of cardiac myocytes.

Calcium channels reconstituted from the skeletal muscle dihydropyridine receptor protein complex and its alpha 1 peptide subunit in lipid bilayers.

In the first part of this study, we show that sDHPR and pDHPR preparations reconstituted into lipid bilayers formed on the tips of patch pipettes exhibit two divalent cation-selective conductance levels of 9 and 20 pS, similar in single-channel conductance to VSCC reported in a variety of intact preparations (see Pelzer et al. and Tsien et al. for review). The larger conductance level is similar to the VSCC identified in intact rat t-tubule membranes and described in sDHPR and pDHPR preparations, and shares many properties in common with activity from L-type VSCC. It is sensitive to augmentation by the DHP agonist (+/-)-BAY K 8644 and cAMP-dependent phosphorylation, and to block by the phenylalkylamine (+/-)-D600 and the inorganic blocker CoCl2. Its open-state probability and open times are increased upon depolarization as expected for a voltage-dependent activation process. Upon depolarization beyond the reversal potential, however, open-state probability and open times decline again. A reasonable way to explain the bell-shaped dependence of open times and open-state probability on membrane potential is to assume voltage-dependent ion-pore interactions that produce closing of the channel at strong negative and positive membrane potentials. By contrast, the smaller conductance level may be similar to the 10.6-pS t-tubule VSCC described by Rosenberg et al. and may best be compared with T-type VSCC. It is largely resistant to augmentation by (+/-)-BAY K 8644 and cAMP-dependent phosphorylation or block by (+/-)-D600, but is sensitive to block by CoCl2. Its open times and open-state probability show a sole dependence on membrane potential where depolarization increases both parameters sigmoidally from close to zero up to a saturating level. Both elementary conductance levels do not exhibit significant inactivation over a wide potential range, which may suggest that skeletal muscle VSCC inactivation is either poorly or not voltage-dependent at all. This possibility seems in agreement with bilayer recordings on reconstituted intact t-tubule membranes and voltage-clamp recordings on intact fibers. It supports the idea that the decline of Ca2+ current in intact skeletal muscle fibers may be due to Ca2+ depletion from the t-tubule system and/or to inactivation induced by Ca2+ release from the sarcoplasmic reticulum. We consistently observe two conductance levels of 9 and 20 pS, either singly, or together in the same bilayer from solubilized DHPR samples and even highly purified DHPR preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of calcium channel function by phosphorylation in guinea pig ventricular cells and phospholipid bilayer membranes.

L-type calcium channel activity of some excitable cells is markedly enhanced by beta-adrenergic agents. The enzymatic cascade underlying this important modulatory effect has been studied with patch-clamp techniques in single dialyzed ventricular cells from guinea pig heart. The steps between the binding of agonist to the beta-receptor and the increase in calcium influx can be summarized as follows: Agonist binding to beta-receptor greater than adenylate cyclase increases greater than cAMP increases greater than cA-kinase increases greater than protein phosphorylation greater than altered calcium channel properties greater than ICa increases A basal phosphorylation reaction seems not to be a prerequisite for calcium channel function. By combining molecular and functional approaches, the purified dihydropyridine-receptor complex from rabbit skeletal muscle transverse-tubules can be reconstituted in phospholipid bilayer membranes to form a functional 20-pS calcium channel that retains the principal regulatory, biochemical, and pharmacologic properties of membrane-bound L-type calcium channels.