Activation of TRPC6 channels is essential for lung ischaemia-reperfusion induced oedema in mice.

Lung ischaemia-reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2(y/-)) or the classical transient receptor potential channel 6 (TRPC6(-/-)) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca(2+) influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2(y/-) cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE.

Moderate calcium channel dysfunction in adult mice with inducible cardiomyocyte-specific excision of the cacnb2 gene.

The major L-type voltage-gated calcium channels in heart consist of an α1C (Ca(V)1.2) subunit usually associated with an auxiliary ß subunit (Ca(V)ß2). In embryonic cardiomyocytes, both the complete and the cardiac myocyte-specific null mutant of Ca(V)ß2 resulted in reduction of L-type calcium currents by up to 75%, compromising heart function and causing defective remodeling of intra- and extra-embryonic blood vessels followed by embryonic death. Here we conditionally excised the Ca(V)ß2 gene (cacnb2) specifically in cardiac myocytes of adult mice (KO). Upon gene deletion, Ca(V)ß2 protein expression declined by >96% in isolated cardiac myocytes and by >74% in protein fractions from heart. These latter protein fractions include Ca(V)ß2 proteins expressed in cardiac fibroblasts. Surprisingly, mice did not show any obvious impairment, although cacnb2 excision was not compensated by expression of other Ca(V)ß proteins or changes of Ca(V)1.2 protein levels. Calcium currents were still dihydropyridine-sensitive, but current density at 0 mV was reduced by <29%. The voltage for half-maximal activation was slightly shifted to more depolarized potentials in KO cardiomyocytes when compared with control cells, but the difference was not significant. In summary, Ca(V)ß2 appears to be a much stronger modulator of L-type calcium currents in embryonic than in adult cardiomyocytes. Although essential for embryonic survival, Ca(V)ß2 down-regulation in cardiomyocytes is well tolerated by the adult mice.

Transient receptor potential melastatin 1 (TRPM1) is an ion-conducting plasma membrane channel inhibited by zinc ions.

TRPM1 is the founding member of the melastatin subgroup of transient receptor potential (TRP) proteins, but it has not yet been firmly established that TRPM1 proteins form ion channels. Consequently, the biophysical and pharmacological properties of these proteins are largely unknown. Here we show that heterologous expression of TRPM1 proteins induces ionic conductances that can be activated by extracellular steroid application. However the current amplitudes observed were too small to enable a reliable biophysical characterization. We overcame this limitation by modifying TRPM1 channels in several independent ways that increased the similarity to the closely related TRPM3 channels. The resulting constructs produced considerably larger currents after overexpression. We also demonstrate that unmodified TRPM1 and TRPM3 proteins form functional heteromultimeric channels. With these approaches, we measured the divalent permeability profile and found that channels containing the pore of TRPM1 are inhibited by extracellular zinc ions at physiological concentrations, in contrast to channels containing only the pore of TRPM3. Applying these findings to pancreatic ß cells, we found that TRPM1 proteins do not play a major role in steroid-activated currents of these cells. The inhibition of TRPM1 by zinc ions is primarily due to a short stretch of seven amino acids present only in the pore region of TRPM1 but not of TRPM3. Combined, our data demonstrate that TRPM1 proteins are bona fide ion-conducting plasma membrane channels. Their distinct biophysical properties allow a reliable identification of endogenous TRPM1-mediated currents.

Increased catecholamine secretion contributes to hypertension in TRPM4-deficient mice.

Hypertension is an underlying risk factor for cardiovascular disease. Despite this, its pathogenesis remains unknown in most cases. Recently, the transient receptor potential (TRP) channel family was associated with the development of several cardiovascular diseases linked to hypertension. The melastatin TRP channels TRPM4 and TRPM5 have distinct properties within the TRP channel family: they form nonselective cation channels activated by intracellular calcium ions. Here we report the identification of TRPM4 proteins in endothelial cells, heart, kidney, and chromaffin cells from the adrenal gland, suggesting that they have a role in the cardiovascular system. Consistent with this hypothesis, Trpm4 gene deletion in mice altered long-term regulation of blood pressure toward hypertensive levels. No changes in locomotor activity, renin-angiotensin system function, electrolyte and fluid balance, vascular contractility, and cardiac contractility under basal conditions were observed. By contrast, inhibition of ganglionic transmission with either hexamethonium or prazosin abolished the difference in blood pressure between Trpm4-/- and wild-type mice. Strikingly, plasma epinephrine concentration as well as urinary excretion of catecholamine metabolites were substantially elevated in Trpm4-/- mice. In freshly isolated chromaffin cells, lack of TRPM4 was shown to cause markedly more acetylcholine-induced exocytotic release events, while neither cytosolic calcium concentration, size, nor density of vesicles were different. We therefore conclude that TRPM4 proteins limit catecholamine release from chromaffin cells and that this contributes to increased sympathetic tone and hypertension.

Quantitative proteomics of the Cav2 channel nano-environments in the mammalian brain.

Local Ca(2+) signaling occurring within nanometers of voltage-gated Ca(2+) (Cav) channels is crucial for CNS function, yet the molecular composition of Cav channel nano-environments is largely unresolved. Here, we used a proteomic strategy combining knockout-controlled multiepitope affinity purifications with high-resolution quantitative MS for comprehensive analysis of the molecular nano-environments of the Cav2 channel family in the whole rodent brain. The analysis shows that Cav2 channels, composed of pore-forming alpha1 and auxiliary beta subunits, are embedded into protein networks that may be assembled from a pool of approximately 200 proteins with distinct abundance, stability of assembly, and preference for the three Cav2 subtypes. The majority of these proteins have not previously been linked to Cav channels; about two-thirds are dedicated to the control of intracellular Ca(2+) concentration, including G protein-coupled receptor-mediated signaling, to activity-dependent cytoskeleton remodeling or Ca(2+)-dependent effector systems that comprise a high portion of the priming and release machinery of synaptic vesicles. The identified protein networks reflect the cellular processes that can be initiated by Cav2 channel activity and define the molecular framework for organization and operation of local Ca(2+) signaling by Cav2 channels in the brain.

Defective survival of naive CD8+ T lymphocytes in the absence of the beta3 regulatory subunit of voltage-gated calcium channels.

The survival of T lymphocytes requires sustained, Ca(2+) influx-dependent gene expression. The molecular mechanism that governs sustained Ca(2+) influx in naive T lymphocytes is unknown. Here we report an essential role for the beta3 regulatory subunit of voltage-gated calcium (Ca(v)) channels in the maintenance of naive CD8(+) T cells. Deficiency in beta3 resulted in a profound survival defect of CD8(+) T cells. This defect correlated with depletion of the pore-forming subunit Ca(v)1.4 and attenuation of T cell antigen receptor (TCR)-mediated global Ca(2+) entry in CD8(+) T cells. Ca(v)1.4 and beta3 associated with T cell signaling machinery and Ca(v)1.4 localized in lipid rafts. Our data demonstrate a mechanism by which Ca(2+) entry is controlled by a Ca(v)1.4-beta3 channel complex in T cells.

Diversity and developmental expression of L-type calcium channel beta2 proteins and their influence on calcium current in murine heart.

By now, little is known on L-type calcium channel (LTCC) subunits expressed in mouse heart. We show that CaVbeta2 proteins are the major CaVbeta components of the LTCC in embryonic and adult mouse heart, but that in embryonic heart CaVbeta3 proteins are also detectable. At least two CaVbeta2 variants of approximately 68 and approximately 72 kDa are expressed. To identify the underlying CaVbeta2 variants, cDNA libraries were constructed from poly(A)(+) RNA isolated from hearts of 7-day-old and adult mice. Screening identified 60 independent CaVbeta2 cDNA clones coding for four types of CaVbeta2 proteins only differing in their 5' sequences. CaVbeta2-N1, -N4, and -N5 but not -N3 were identified in isolated cardiomyocytes by RT-PCR and were sufficient to reconstitute the CaVbeta2 protein pattern in vitro. Significant L-type Ca(2+) currents (I(Ca)) were recorded in HEK293 cells after co-expression of CaV1.2 and CaVbeta2. Current kinetics were determined by the type of CaVbeta2 protein, with the approximately 72-kDa CaVbeta2a-N1 shifting the activation of I(Ca) significantly to depolarizing potentials compared with the other CaVbeta2 variants. Inactivation of I(Ca) was accelerated by CaVbeta2a-N1 and -N4, which also lead to slower activation compared with CaVbeta2a-N3 and -N5. In summary, this study reveals the molecular LTCC composition in mouse heart and indicates that expression of various CaVbeta2 proteins may be used to adapt the properties of LTCCs to changing myocardial requirements during development and that CaVbeta2a-N1-induced changes of I(Ca) kinetics might be essential in embryonic heart.

Transgenic simulation of human heart failure-like L-type Ca2+-channels: implications for fibrosis and heart rate in mice.

AIMS: Cardiac L-type Ca(2+)-currents show distinct alterations in chronic heart failure, including increased single-channel activity and blunted adrenergic stimulation, but minor changes of whole-cell currents. Expression of L-type Ca(2+)-channel beta(2)-subunits is enhanced in human failing hearts. In order to determine whether prolonged alteration of Ca(2+)-channel gating by beta(2)-subunits contributes to heart failure pathogenesis, we generated and characterized transgenic mice with cardiac overexpression of a beta(2a)-subunit or the pore Ca(v)1.2 or both, respectively. METHODS AND RESULTS: Four weeks induction of cardiac-specific overexpression of rat beta(2a)-subunits shifted steady-state activation and inactivation of whole-cell currents towards more negative potentials, leading to increased Ca(2+)-current density at more negative test potentials. Activity of single Ca(2+)-channels was increased in myocytes isolated from beta(2a)-transgenic mice. Ca(2+)-current stimulation by 8-Br-cAMP and okadaic acid was blunted in beta(2a)-transgenic myocytes. In vivo investigation revealed hypotension and bradycardia upon Ca(v)1.2-transgene expression but not in mice only overexpressing beta(2a). Double-transgenics showed cardiac arrhythmia. Interstitial fibrosis was aggravated by the beta(2a)-transgene compared with Ca(v)1.2-transgene expression alone. Overt cardiac hypertrophy was not observed in any model. CONCLUSION: Cardiac overexpression of a Ca(2+)-channel beta(2a)-subunit alone is sufficient to induce Ca(2+)-channel properties characteristic of chronic human heart failure. beta(2a)-overexpression by itself did not induce cardiac hypertrophy or contractile dysfunction, but aggravated the development of arrhythmia and fibrosis in Ca(v)1.2-transgenic mice.

Ca2+ channel currents and contraction in CaVbeta3-deficient ileum smooth muscle from mouse.

Voltage activated L-type Ca(2+) channels are the principal Ca(2+) channels in intestinal smooth muscle cells. They comprise the ion conducting Ca(V)1 pore and the ancillary subunits alpha(2)delta and beta. Of the four Ca(V)beta subunits Ca(V)beta(3) is assumed to be the relevant Ca(V)beta protein in smooth muscle. In protein lysates isolated from mouse ileum longitudinal smooth muscle we could identify the Ca(V)1.2, Ca(V)alpha(2), Ca(V)beta(2) and Ca(V)beta(3) proteins, but not the Ca(V)beta(1) and Ca(V)beta(4) proteins. Protein levels of Ca(V)1.2, Ca(V)alpha(2) and Ca(V)beta(2) are not altered in ileum smooth muscle obtained from Ca(V)beta(3)-deficient mice indicating that there is no compensatory increase of the expression of these channel proteins. Neither the Ca(V)beta(2) nor the other Ca(V)beta proteins appear to substitute for the lacking Ca(V)beta(3). L-type Ca(2+) channel properties including current density, inactivation kinetics as well as Cd(2+)- and dihydropyridine sensitivity were identical in cells of both genotypes suggesting that they do not require the presence of a Ca(V)beta(3) protein. However, a key hallmark of the Ca(V)beta modulation of Ca(2+) current, the hyperpolarisation of channel activation is slightly but significantly reduced by 4 mV. In addition to L-type Ca(2+) currents T-type Ca(2+) currents could be recorded in the murine ileum smooth muscle cells, but T-type currents were not affected by the lack of Ca(V)beta(3). Both proteins, Ca(V)beta(2) and Ca(V)beta(3) are localized near the plasma membrane and the localization of Ca(V)beta(2) is not altered in Ca(V)beta(3) deficient cells. Spontaneous contractions and potassium and carbachol induced contractions are not significantly different between ileum longitudinal smooth muscle strips from mice of both genotypes. In summary the data show that in ileum smooth muscle cells, Ca(V)beta(3) has only subtle effects on L-type Ca(2+) currents, appears not to be required for spontaneous and potassium induced contraction but might have a function beyond being a Ca(2+) channel subunit.

Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel TRPM4.

Mast cells are key effector cells in allergic reactions. Aggregation of the receptor FcepsilonRI in mast cells triggers the influx of calcium (Ca(2+)) and the release of inflammatory mediators. Here we show that transient receptor potential TRPM4 proteins acted as calcium-activated nonselective cation channels and critically determined the driving force for Ca(2+) influx in mast cells. Trpm4(-/-) bone marrow-derived mast cells had more Ca(2+) entry than did TRPM4(+/+) cells after FcepsilonRI stimulation. Consequently, Trpm4(-/-) bone marrow-derived mast cells had augmented degranulation and released more histamine, leukotrienes and tumor necrosis factor. Trpm4(-/-) mice had a more severe IgE-mediated acute passive cutaneous anaphylactic response, whereas late-phase passive cutaneous anaphylaxis was not affected. Our results establish the physiological function of TRPM4 channels as critical regulators of Ca(2+) entry in mast cells.

Specific detection and semi-quantitative analysis of TRPC4 protein expression by antibodies.

In mouse tissues two variants of the transient receptor potential (canonical) (TRPC) 4 protein are expressed: the "full-length" TRPC4 protein and a slightly smaller variant, called TRPC4Delta(761-864), which lacks 84 amino acid residues. Although the presence of mRNA encoding the TRPC4 protein in mammalian cells and the detection of the heterologously expressed TRPC4 protein by Western blot analysis have been reported, the unequivocal detection of endogenous TRPC4 proteins has proven difficult. In the present study we compared polyclonal antibodies for the detection of TRPC4 proteins in mouse tissues and monitored their specificity and reliability by analysing corresponding tissues from TRPC4-deficient mice. In addition we introduced a procedure that allows us to estimate the amount of TRPC4 protein expressed in a single cell. Using this technique it appears that the amount of TRPC4 protein expressed stably in HEK 293 cells is at least fourfold higher than the amount of TRPC4 protein expressed endogenously in the bovine adrenocortical cell line SBAC.