Dendritic cell (DC) maturation and migration are events critical for the initiation of immune responses. After encountering pathogens, DCs upregulate the expression of costimulatory molecules and subsequently migrate to secondary lymphoid organs. Calcium (Ca2+) entry governs the functions of many hematopoietic cell types, but the role of Ca2+ entry in DC biology remains unclear. Here we report that the Ca2+-activated nonselective cation channel TRPM4 was expressed in and controlled the Ca2+ homeostasis of mouse DCs. The absence of TRPM4, which elicited Ca2+ overload, did not influence DC maturation but did considerably impair chemokine-dependent DC migration. Our results establish TRPM4-regulated Ca2+ homeostasis as crucial for DC mobility but not maturation and emphasize that DC maturation and migration are independently regulated.

Background and purpose: TRPM4 and TRPM5 are calcium-activated non-selective cation channels with almost identical characteristics. TRPM4 is detected in several tissues including heart, kidney, brainstem, cerebral artery and immune system whereas TRPM5 expression is more restricted. Determination of their roles in physiological processes requires specific pharmacological tools. TRPM4 is inhibited by glibenclamide, a modulator of ATP binding cassette proteins (ABC transporters), such as the cystic fibrosis transmembrane conductance regulator (CFTR). We took advantage of this similarity to investigate the effect of hydroxytricyclic compounds shown to modulate ABC transporters, on TRPM4 and TRPM5. Experimental approach: Experiments were conducted using HEK-293 cells permanently transfected to express human TRPM4 or TRPM5. Currents were recorded using the whole-cell and inside-out variants of the patch-clamp technique. Key results: The CFTR channel activator benzo[c]quinolizinium MPB-104 inhibited TRPM4 current with an IC 50 in the range of 2 Â 10 À5 M, with no effect on single-channel conductance. In addition, 9-phenanthrol, lacking the chemical groups necessary for CFTR activation, also reversibly inhibited TRPM4 with a similar IC 50 . Channel inhibition was voltage independent. The IC 50 determined in the whole-cell and inside-out experiments were similar, suggesting a direct effect of the molecule. However, 9-phenanthrol was ineffective on TRPM5, the most closely related channel within the TRP protein family. Conclusions and implications: We identify 9-phenanthrol as a TRPM4 inhibitor, without effects on TRPM5. It could be valuable in investigating the physiological functions of TRPM4, as distinct from those of TRPM5.

TRPM4 is functionally expressed in SAN cells and may be a key player in the generation and/or perturbation of heart rhythm.

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current (I ti Channel activity was reduced in the presence of 0.5 mM ATP or 10 µM glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 mM flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC Ca channel is a serious candidate to support the delayed after-depolarizations observed in [Ca 2+ ] overload and thus may be implicated in the genesis of arrhythmias.

Cardiac hypertrophy is associated with electrophysiological modifications, including modification of action potential shape that can give rise to arrhythmias. We report here a higher detection of a calcium-activated nonselective cation current in cardiomyocytes of spontaneously hypertensive rats (SHRs), a model of hypertension and heart hypertrophy when compared with Wistar-Kyoto (WKY) rat, its normotensive equivalent. Freshly isolated cells from the left ventricles of 3- to 6-month-old WKY rats or SHRs were used for patch-clamp recordings. In inside-out patches, the channel presented a linear conductance of 25+/-0.5 pS, did not discriminate Na(+) over K(+), and was not permeable to Ca(2+). Open probability was increased by depolarization and a rise in [Ca(2+)](i) (dissociation constant=10+/-5.4 micromol/L) but reduced by 0.5 mmol/L [ATP](i), 10 micromol/L glibenclamide, or flufenamic acid (IC(50)=5.5+/-1.7 micromol/L). Thus, it owns the fingerprint of the TRPM4 current. Although rarely detected in WKY cardiomyocytes, the current was present in >50% of patches from SHR cardiomyocytes. Moreover, by performing RT-PCR from ventricular samples, we observed that TRPM4 mRNA detection was higher in SHRs than in WKY rats. We propose that a TRPM4 current is expressed in ventricular cardiomyocytes from SHRs. According to its properties, this channel may contribute to the transient inward current implicated in delayed-after-depolarizations observed during [Ca(2+)] overload of cardiomyocytes.

Aims/hypothesis Mitochondria-associated endoplasmic reticulum membranes (MAMs) are regions of the endoplasmic reticulum (ER) tethered to mitochondria and controlling calcium (Ca 2+ ) transfer between both organelles through the complex formed between the voltage-dependent anion channel, glucose-regulated protein 75 and inositol 1,4,5-triphosphate receptor (IP3R). We recently identified cyclophilin D (CYPD) as a new partner of this complex and demonstrated a new role for MAMs in the control of insulin's action in the liver. Here, we report on the mechanisms by which disruption of MAM integrity induces hepatic insulin resistance in CypD (also known as Ppif)-knockout (KO) mice. Methods We used either in vitro pharmacological and genetic inhibition of CYPD in HuH7 cells or in vivo loss of CYPD in mice to investigate ER-mitochondria interactions, inter-organelle Ca 2+ exchange, organelle homeostasis and insulin action. Results Pharmacological and genetic inhibition of CYPD concomitantly reduced ER-mitochondria interactions, inhibited inter-organelle Ca 2+ exchange, induced ER stress and altered insulin signalling in HuH7 cells. In addition, histaminestimulated Ca 2+ transfer from ER to mitochondria was blunted in isolated hepatocytes of CypD-KO mice and this was associated with an increase in ER calcium store. Interestingly, disruption of inter-organelle Ca 2+ transfer was associated with ER stress, mitochondrial dysfunction, lipid accumulation, activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)ε and insulin resistance in liver of CypD-KO mice. Finally, CYPD-related alterations of insulin signalling were mediated by activation of PKCε rather than JNK in HuH7 cells. Conclusions/interpretation Disruption of IP3R-mediated Ca 2+ signalling in the liver of CypD-KO mice leads to hepatic insulin resistance through disruption of organelle interaction and function, increase in lipid accumulation and activation of PKCε. Modulation of ER-mitochondria Ca 2+ exchange may thus provide an exciting new avenue for treating hepatic insulin resistance.

RationaleTRPM4 is a non-selective Ca2+-activated cation channel expressed in the heart, particularly in the atria or conduction tissue. Mutations in the Trpm4 gene were recently associated with several human conduction disorders such as Brugada syndrome. TRPM4 channel has also been implicated at the ventricular level, in inotropism or in arrhythmia genesis due to stresses such as ß-adrenergic stimulation, ischemia-reperfusion, and hypoxia re-oxygenation. However, the physiological role of the TRPM4 channel in the healthy heart remains unclear.ObjectivesWe aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4 -/-) model.Methods and ResultsMorpho-functional analysis revealed left ventricular (LV) eccentric hypertrophy in Trpm4 -/- mice, with an increase in both wall thickness and chamber size in the adult mouse (aged 32 weeks) when compared to Trpm4+/+ littermate controls. Immunofluorescence on frozen heart cryosections and qPCR analysis showed no fibrosis or cellular hypertrophy. Instead, cardiomyocytes in Trpm4-/- mice were smaller than Trpm4+/+with a higher density. Immunofluorescent labeling for phospho-histone H3, a mitosis marker, showed that the number of mitotic myocytes was increased 3-fold in the Trpm4-/-neonatal stage, suggesting hyperplasia. Adult Trpm4 -/- mice presented multilevel conduction blocks, as attested by PR and QRS lengthening in surface ECGs and confirmed by intracardiac exploration. Trpm4-/-mice also exhibited Luciani-Wenckebach atrioventricular blocks, which were reduced following atropine infusion, suggesting paroxysmal parasympathetic overdrive. In addition, Trpm4 -/- mice exhibited shorter action potentials in atrial cells. This shortening was unrelated to modifications of the voltage-gated Ca2+ or K+ currents involved in the repolarizing phase.ConclusionsTRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development.

Calcium-activated nonselective cationic currents have been known for 30 years, but their physiological implications have remained unresolved until the recent cloning of the TRPM4 ion channel. Since then, TRPM4 has been identified as a key modulator of numerous calcium-dependent mechanisms such as the immune response, insulin secretion, cerebral artery constriction, respiratory rhythm, and cardiac conduction.