Johannes Oberwinkler

Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic beta cells.

Transient receptor potential (TRP) cation channels are renowned for their ability to sense diverse chemical stimuli. Still, for many members of this large and heterogeneous protein family it is unclear how their activity is regulated and whether they are influenced by endogenous substances. On the other hand, steroidal compounds are increasingly recognized to have rapid effects on membrane surface receptors that often have not been identified at the molecular level. We show here that TRPM3, a divalent-permeable cation channel, is rapidly and reversibly activated by extracellular pregnenolone sulphate, a neuroactive steroid. We show that pregnenolone sulphate activates endogenous TRPM3 channels in insulin-producing β cells. Application of pregnenolone sulphate led to a rapid calcium influx and enhanced insulin secretion from pancreatic islets. Our results establish that TRPM3 is an essential component of an ionotropic steroid receptor enabling unanticipated crosstalk between steroidal and insulin-signalling endocrine systems.

Light Activation, Adaptation, and Cell Survival Functions of the Na+/Ca2+ Exchanger CalX

In sensory neurons, Ca(2+) entry is crucial for both activation and subsequent attenuation of signaling. Influx of Ca(2+) is counterbalanced by Ca(2+) extrusion, and Na(+)/Ca(2+) exchange is the primary mode for rapid Ca(2+) removal during and after sensory stimulation. However, the consequences on sensory signaling resulting from mutations in Na(+)/Ca(2+) exchangers have not been described. Here, we report that mutations in the Drosophila Na(+)/Ca(2+) exchanger calx have a profound effect on activity-dependent survival of photoreceptor cells. Loss of CalX activity resulted in a transient response to light, a dramatic decrease in signal amplification, and unusually rapid adaptation. Conversely, overexpression of CalX had reciprocal effects and greatly suppressed the retinal degeneration caused by constitutive activity of the TRP channel. These results illustrate the critical role of Ca(2+) for proper signaling and provide genetic evidence that Ca(2+) overload is responsible for a form of retinal degeneration resulting from defects in the TRP channel.

Bile Acids Acutely Stimulate Insulin Secretion of Mouse β-Cells via Farnesoid X Receptor Activation and KATP Channel Inhibition

Type 2 diabetes mellitus is associated with alterations in bile acid (BA) signaling. The aim of our study was to test whether pancreatic β-cells contribute to BA-dependent regulation of glucose homeostasis. Experiments were performed with islets from wild-type, farnesoid X receptor (FXR) knockout (KO), and β-cell ATP-dependent K+ (KATP) channel gene SUR1 (ABCC8) KO mice, respectively. Sodium taurochenodeoxycholate (TCDC) increased glucose-induced insulin secretion. This effect was mimicked by the FXR agonist GW4064 and suppressed by the FXR antagonist guggulsterone. TCDC and GW4064 stimulated the electrical activity of β-cells and enhanced cytosolic Ca2+ concentration ([Ca2+]c). These effects were blunted by guggulsterone. Sodium ursodeoxycholate, which has a much lower affinity to FXR than TCDC, had no effect on [Ca2+]c and insulin secretion. FXR activation by TCDC is suggested to inhibit KATP current. The decline in KATP channel activity by TCDC was only observed in β-cells with intact metabolism and was reversed by guggulsterone. TCDC did not alter insulin secretion in islets of SUR1-KO or FXR-KO mice. TCDC did not change islet cell apoptosis. This is the first study showing an acute action of BA on β-cell function. The effect is mediated by FXR by nongenomic elements, suggesting a novel link between FXR activation and KATP channel inhibition.

Mechanisms of Light Adaptation in Drosophila Photoreceptors

Phototransduction in Drosophila is mediated by a phospholipase C (PLC) cascade culminating in activation of transient receptor potential (TRP) channels. Ca(2+) influx via these channels is required for light adaptation, but although several molecular targets of Ca(2+)-dependent feedback have been identified, their contribution to adaptation is unclear. By manipulating cytosolic Ca(2+) via the Na(+)/Ca(2+) exchange equilibrium, we found that Ca(2+) inhibited the light-induced current (LIC) over a range corresponding to steady-state light-adapted Ca(2+) levels (0.1-10 microM Ca(2+)) and accurately mimicked light adaptation. However, PLC activity monitored with genetically targeted PIP(2)-sensitive ion channels (Kir2.1) was first inhibited by much higher (>/= approximately 50 microM) Ca(2+) levels, which occur only transiently in vivo. Ca(2+)-dependent inhibition of PLC, but not the LIC, was impaired in mutants (inaC) of protein kinase C (PKC). The results indicate that light adaptation is primarily mediated downstream of PLC and independently of PKC by Ca(2+)-dependent inhibition of TRP channels. This is interpreted as a strategy to prevent inhibition of PLC by global steady-state light-adapted Ca(2+) levels, whereas rapid inhibition of PLC by local Ca(2+) transients is required to terminate the response and ensures that PIP(2) reserves are not depleted during stimulation.

Ultraviolet vision: The colourful world of the mantis shrimp

Humans cannot see ultraviolet light, but many arthropods and vertebrates can because they have a single photo-receptor with a peak sensitivity to light at wavelengths of around 350 nanometres (ref. 1). Here we use electrophysiological methods to investigate the vision of the mantis shrimp, Neogonodactylus oerstedii. We find that this marine crustacean has at least four types of photoreceptor for ultraviolet light that are located in cells of the eye known as R8 cells. These photoreceptors are maximally sensitive to light of wavelengths 315, 330, 340 and 380 nm. Together with previous evidence, this finding indicates that the remarkable colour-vision system in these stomatopod crustaceans may be unique, as befits their habitat of kaleidoscopically colourful tropical coral reefs.

The colourful world of the mantis shrimp

Humans cannot see ultraviolet light, but many arthropods and vertebrates can because they have a single photo-receptor with a peak sensitivity to light at wavelengths of around 350 nanometres (ref. 1). Here we use electrophysiological methods to investigate the vision of the mantis shrimp, Neogonodactylus oerstedii. We find that this marine crustacean has at least four types of photoreceptor for ultraviolet light that are located in cells of the eye known as R8 cells. These photoreceptors are maximally sensitive to light of wavelengths 315, 330, 340 and 380 nm. Together with previous evidence, this finding indicates that the remarkable colour-vision system in these stomatopod crustaceans may be unique, as befits their habitat of kaleidoscopically colourful tropical coral reefs.

TRPM3 channels provide a regulated influx pathway for zinc in pancreatic beta cells.

Zinc is stored in insulin-containing dense core vesicles of pancreatic β-cells where it forms crystals together with insulin and calcium ions. Zinc ions are therefore released together with insulin upon exocytosis of these vesicles. Consequently, pancreatic β-cells need to take up large amounts of zinc from the extracellular space across their plasma membrane. The pathways for zinc uptake are only partially understood. TRPM3 channels are present in pancreatic β-cells and can be activated by the endogenous steroid pregnenolone sulfate. We demonstrate here that recombinant TRPM3 channels are highly permeable for many divalent cations, in particular also for zinc ions. Importantly, TRPM3 channels endogenously expressed in pancreatic β-cells are also highly permeable for zinc ions. Using FluoZin3 to image changes of the intracellular zinc concentration, we show that pancreatic β-cells take up zinc through TRPM3 channels even when extracellular zinc concentrations are low and physiological levels of calcium and magnesium are present. Activation of TRPM3 channels also leads to depolarization of β-cells and to additional zinc influx through voltage-gated calcium channels. Our data establish that TRPM3 channels constitute a regulated entry pathway for zinc ions in pancreatic β-cells.

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 β 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.

Characterization of a proton-activated, outwardly rectifying anion channel.

Anion channels are present in every mammalian cell and serve many different functions, including cell volume regulation, ion transport across epithelia, regulation of membrane potential and vesicular acidification. Here we characterize a proton‐activated, outwardly rectifying current endogenously expressed in HEK293 cells. Binding of three to four protons activated the anion permeable channels at external pH below 5.5 (50% activation at pH 5.1). The proton‐activated current is strongly outwardly rectifying, due to an outwardly rectifying single channel conductance and an additional voltage dependent facilitation at depolarized membrane potentials. The anion channel blocker 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonic acid (DIDS) rapidly and potently inhibited the channel (IC50: 2.9 μm). Flufenamic acid blocked this channel only slowly, while mibefradil and amiloride at high concentrations had no effect. As determined from reversal potential measurements under bi‐ionic conditions, the relative permeability sequence of this channel was SCN−> I−> NO3−> Br−> Cl−. None of the previously characterized anion channel matches the properties of the proton‐activated, outwardly rectifying channel. Specifically, the proton‐activated and the volume‐regulated anion channels are two distinct and separable populations of ion channels, each having its own set of biophysical and pharmacological properties. We also demonstrate endogenous proton‐activated currents in primary cultured hippocampal astrocytes. The proton‐activated current in astrocytes is also carried by anions, strongly outwardly rectifying, voltage dependent and inhibited by DIDS. Proton‐activated, outwardly rectifying anion channels therefore may be a broadly expressed part of the anionic channel repertoire of mammalian cells.

Does Ca2+ Reach Millimolar Concentrations after Single Photon Absorption in Drosophila Photoreceptor Microvilli?

The quantum bump, the elementary event of fly phototransduction induced by the absorption of a single photon, is a small, transient current due to the opening of cation-channels permeable to Ca2+. These channels are located in small, tube-like protrusions of the cell membrane, the microvilli. Using a modeling approach, we calculate the changes of free Ca2+ concentration inside the microvilli, taking into account influx and diffusion of Ca2+. Independent of permeability ratios and Ca2+ buffering, we find that the free Ca2+ concentrations rise to millimolar values, as long as we assume that all activated channels are located in a single microvillus. When we assume that as much as 25 microvilli participate in a single bump, the free Ca2+ concentration still reaches values higher than 80 microM. These very high concentrations show that the microvilli of fly photoreceptors are unique structures in which the Ca2+ signaling is even more extreme than in calcium concentration microdomains very close to Ca2+ channels.