Proteins of the mammalian TRP (transient receptor potential) family form a heterogenous group of cation channels important for cellular Ca²⁺ signaling and homeostasis. Here we present the full-length sequence of TRPM3, a member of the melastatin-like subfamily (TRPM) of TRP channels. TRPM3 expression was found in human kidney and brain. HEK293 cells transiently transfected with TRPM3 showed a constitutive Ca²⁺ and Mn²⁺ entry. Whole-cell patch clamp experiments confirmed the spontaneous activity of TRPM3 and revealed permeability ratios P_Ca/P_Na of 1.57 and P_Na/P_Cs of 0.75. In cell-attached patches, spontaneous inward and outward currents were observed. At negative membrane potentials and in the presence of either 140 mm Cs⁺, 140 mm Na⁺, or 100 mm Ca²⁺ in the pipette solution, the single channel conductance levels were 133, 83, and 65 pS, respectively. The Ca²⁺ entry in TRPM3-expressing HEK293 cells increased during treatment with hypotonic extracellular solution. The reduction of extracellular osmolarity was accompanied by cell swelling, suggesting volume-regulated activity of TRPM3. From its function and expression in human kidney, we propose a role of TRPM3 in renal Ca²⁺ homeostasis.

Excessive cytosolic calcium ion (Ca²⁺) accumulation during cerebral ischemia triggers neuronal cell death, but the underlying mechanisms are poorly understood. Capacitive Ca²⁺ entry (CCE) is a process whereby depletion of intracellular Ca²⁺ stores causes the activation of plasma membrane Ca²⁺ channels. In nonexcitable cells, CCE is controlled by the endoplasmic reticulum (ER)–resident Ca²⁺ sensor STIM1, whereas the closely related protein STIM2 has been proposed to regulate basal cytosolic and ER Ca²⁺ concentrations and make only a minor contribution to CCE. Here, we show that STIM2, but not STIM1, is essential for CCE and ischemia-induced cytosolic Ca²⁺ accumulation in neurons. Neurons from Stim2^−/− mice showed significantly increased survival under hypoxic conditions compared to neurons from wild-type controls both in culture and in acute hippocampal slice preparations. In vivo, Stim2^−/− mice were markedly protected from neurological damage in a model of focal cerebral ischemia. These results implicate CCE in ischemic neuronal cell death and establish STIM2 as a critical mediator of this process.

Microglial cells are the host macrophages in the central nervous system and respond to brain injury and various neurological diseases. In this process, microglial cells undergo multiple morphological and functional changes from the resting cell toward a fully activated, phagocyting tissue macrophage. In culture, bacterial lipopolysaccharide (LPS) is a frequently used tool to induce this activation. By using calcium-imaging and patch-clamp techniques, we investigated the effect of hydrogen peroxide (H₂O₂), which is released by macrophagic cells themselves, on the intracellular calcium concentration and ion currents in cultured rat microglia. Application of 0.1–5 mM H₂O₂ for several minutes induced small responses in untreated cells but a large calcium influx and cation current in LPS-treated cells. In both untreated and LPS-treated microglia, internal perfusion of ADP-ribose (ADPR) via the patch pipette elicited large cation currents. Both stimuli, H₂O₂ and ADPR, have been reported to activate the recently cloned nonselective cation channel TRPM2. RT-PCR analysis from cultured rat glial and neuronal cells confirmed a strong expression of TRPM2 in rat microglia but not in astrocytes and cerebellar granule cells. In situ hybridizations from mouse brain showed a distribution of TRPM2, which is compatible with the expression in microglial cells. In conclusion, we describe here a novel calcium influx pathway in microglia coupled to hydrogen peroxide and ADPR and provide evidence that this pathway involves TRPM2. The increased sensitivity to H₂O₂ in LPS-stimulated cells suggests a role for TRPM2 in the calcium signaling of activated microglia.

TRPM2 is a Ca²⁺‐permeable nonselective cation channel activated by intracellular ADP‐ribose (ADPR) and by hydrogen peroxide (H₂O₂). We investigated the modulation of TRPM2 activity by N‐(p‐amylcinnamoyl)anthranilic acid (ACA). ACA has previously been reported to inhibit phospholipase A₂ (PLA₂). Using patch‐clamp and calcium‐imaging techniques, we show that extracellular application of 20 μM ACA completely blocked ADPR‐induced whole‐cell currents and H₂O₂‐induced Ca²⁺ signals (IC₅₀=1.7 μM) in HEK293 cells transfected with human TRPM2. Two other PLA₂ inhibitors, p‐bromophenacyl bromide (BPB; 100 μM) and arachidonyl trifluoromethyl ketone (20 μM), had no significant effect on ADPR‐stimulated TRPM2 activity. Inhibition of TRPM2 whole‐cell currents by ACA was voltage independent and accelerated at decreased pH. ACA was ineffective when applied intracellularly. The single‐channel conductance was not changed during ACA treatment, suggesting a reduction of TRPM2 open probability by modulating channel gating. ACA (20 μM) also blocked currents through human TRPM8 and TRPC6 expressed in HEK293 cells, while BPB (100 μM) was ineffective. TRPC6‐mediated currents (IC₅₀=2.3 μM) and TRPM8‐induced Ca²⁺ signals (IC₅₀=3.9 μM) were blocked in a concentration‐dependent manner. ADPR‐induced currents in human U937 cells, endogeneously expressing TRPM2 protein, were fully suppressed by 20 μM ACA. Our data indicate that ACA modulates the activity of different TRP channels independent of PLA₂ inhibition. Owing to its high potency and efficacy ACA can serve, in combination with other blockers, as a useful tool for studying the unknown function of TRPM2 in native cells. British Journal of Pharmacology (2006) 148, 264–273. doi:10.1038/sj.bjp.0706739

TRPM3, a member of the melastatin-like transient receptor potential channel subfamily (TRPM), is predominantly expressed in human kidney and brain. TRPM3 mediates spontaneous Ca²⁺ entry and nonselective cation currents in transiently transfected human embryonic kidney 293 cells. Using measurements with the Ca²⁺-sensitive fluorescent dye fura-2 and the whole-cell patch-clamp technique, we found that d-erythro-sphingosine, a metabolite arising during the de novo synthesis of cellular sphingolipids, activated TRPM3. Other transient receptor potential (TRP) channels tested [classic or canonical TRP (TRPC3, TRPC4, TRPC5), vanilloid-like TRP (TRPV4, TRPV5, TRPV6), and melastatin-like TRP (TRPM2)] did not significantly respond to application of sphingosine. Sphingosine-induced TRPM3 activation was not mediated by inhibition of protein kinase C, depletion of intracellular Ca²⁺ stores, and intracellular conversion of sphingosine to sphingosine-1-phosphate. Although sphingosine-1-phosphate and ceramides had no effect, two structural analogs of sphingosine, dihydro-d-erythro-sphingosine and N,N-dimethyl-d-erythro-sphingosine, also activated TRPM3. Sphingolipids, including sphingosine, are known to have inhibitory effects on a variety of ion channels. Thus, TRPM3 is the first ion channel activated by sphingolipids.