Structural requirements of steroidal agonists of transient receptor potential melastatin 3 (TRPM3) cation channels.

BACKGROUND AND PURPOSE: Transient receptor potential melastatin 3 (TRPM3) proteins form non-selective but calcium-permeable membrane channels, rapidly activated by extracellular application of the steroid pregnenolone sulphate and the dihydropyridine nifedipine. Our aim was to characterize the steroid binding site by analysing the structural chemical requirements for TRPM3 activation. EXPERIMENTAL APPROACH: Whole-cell patch-clamp recordings and measurements of intracellular calcium concentrations were performed on HEK293 cells transfected with TRPM3 (or untransfected controls) during superfusion with pharmacological substances. KEY RESULTS: Pregnenolone sulphate and nifedipine activated TRPM3 channels supra-additively over a wide concentration range. Other dihydropyridines inhibited TRPM3 channels. The natural enantiomer of pregnenolone sulphate was more efficient in activating TRPM3 channels than its synthetic mirror image. However, both enantiomers exerted very similar inhibitory effects on proton-activated outwardly rectifying anion channels. Epiallopregnanolone sulphate activated TRPM3 almost equally as well as pregnenolone sulphate. Exchanging the sulphate for other chemical moieties showed that a negative charge at this position is required for activating TRPM3 channels. CONCLUSIONS AND IMPLICATIONS: Our data demonstrate that nifedipine and pregnenolone sulphate act at different binding sites when activating TRPM3. The latter activates TRPM3 by binding to a chiral and thus proteinaceous binding site, as inferred from the differential effects of the enantiomers. The double bond between position C5 and C6 of pregnenolone sulphate is not strictly necessary for the activation of TRPM3 channels, but a negative charge at position C3 of the steroid is highly important. These results provide a solid basis for understanding mechanistically the rapid chemical activation of TRPM3 channels.

Citrus fruit and fabacea secondary metabolites potently and selectively block TRPM3.

BACKGROUND AND PURPOSE: The melastatin-related transient receptor potential TRPM3 is a calcium-permeable nonselective cation channel that can be activated by the neurosteroid pregnenolone sulphate (PregS) and heat. TRPM3-deficient mice show an impaired perception of noxious heat. Hence, drugs inhibiting TRPM3 possibly get in focus of analgesic therapy. EXPERIMENTAL APPROACH: Fluorometric methods were used to identify novel TRPM3-blocking compounds and to characterize their potency and selectivity to block TRPM3 but not other sensory TRP channels. Biophysical properties of the block were assessed using electrophysiological methods. Single cell calcium measurements confirmed the block of endogenously expressed TRPM3 channels in rat and mouse dorsal root ganglion (DRG) neurones. KEY RESULTS: By screening a compound library, we identified three natural compounds as potent blockers of TRPM3. Naringenin and hesperetin belong to the citrus fruit flavanones, and ononetin is a deoxybenzoin. Eriodictyol, a metabolite of naringenin and hesperetin, was still biologically active as a TRPM3 blocker. The compounds exhibited a marked specificity for recombinant TRPM3 and blocked PregS-induced [Ca(2+)]i signals in freshly isolated DRG neurones. CONCLUSION AND IMPLICATIONS: The data indicate that citrus fruit flavonoids are potent and selective blockers of TRPM3. Their potencies ranged from upper nanomolar to lower micromolar concentrations. Since physiological functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo. Considering the involvement of TRPM3 in nociception, TRPM3 blockers may represent a novel concept for analgesic treatment.

TRPM3.

TRPM3 is the last identified member of the TRPM subfamily and is most closely related to TRPM1. Due to alternative splicing, the TRPM3 gene encodes a large number of different variants. One splice event, affecting the pore-forming region of the channel, changes its selectivity for divalent cations. In this review, we give an overview of the identified TRPM3 variants and compare their functional properties.

TRPM3, a biophysical enigma?

TRPM3 [TRP (transient receptor potential) melastatin 3] is one of the least investigated proteins of the TRP family of ion channels. Heterologously expressed TRPM3 channels are constitutively active, have an outwardly rectifying current-voltage relationship and are inhibited by intracellular Mg(2+) ions. Besides these rather common features, in which TRPM3 channels resemble the closely related channels TRPM6 and TRPM7, TRPM3 channels have several unique characteristics. The TRPM3 gene encodes a plethora of different proteins owing to alternative splicing and alternative exon usage. One site of alternative splicing affects the ion-conducting pore region and profoundly alters the pore properties of the encoded channels. The channels having the longer pore region efficiently conduct univalent cations, but are only poorly permeated by bivalent cations. Conversely, the channels with the shorter pore region are highly permeable to bivalent cations. Unusually, the short-pore TRPM3 channels are inhibited by extracellular Na(+) ions. At physiological sodium concentration, this block is very strong, making it difficult to envision a physiological function for these ion channels. Recently, pharmacological investigations have been initiated in order to identify substances that influence TRPM3 channel activity. With the use of such substances, it might be possible to identify TRPM3 channels in their native environment and to elucidate some of their physiological roles. Hopefully, TRPM3 channels will then no longer appear to be as enigmatic as they do right now.

Calcium imaging demonstrates colocalization of calcium influx and extrusion in fly photoreceptors.

During illumination, Ca(2+) enters fly photoreceptor cells through light-activated channels that are located in the rhabdomere, the compartment specialized for phototransduction. From the rhabdomere, Ca(2+) diffuses into the cell body. We visualize this process by rapidly imaging the fluorescence in a cross section of a photoreceptor cell injected with a fluorescent Ca(2+) indicator in vivo. The free Ca(2+) concentration in the rhabdomere shows a very fast and large transient shortly after light onset. The free Ca(2+) concentration in the cell body rises more slowly and displays a much smaller transient. After approximately 400 ms of light stimulation, the Ca(2+) concentration in both compartments reaches a steady state, indicating that thereafter an amount of Ca(2+), equivalent to the amount of Ca(2+) flowing into the cell, is extruded. Quantitative analysis demonstrates that during the steady state, the free Ca(2+) concentration in the rhabdomere and throughout the cell body is the same. This shows that Ca(2+) extrusion takes place very close to the location of Ca(2+) influx, the rhabdomere, because otherwise gradients in the steady-state distribution of Ca(2+) should be measured. The close colocalization of Ca(2+) influx and Ca(2+) extrusion ensures that, after turning off the light, Ca(2+) removal from the rhabdomere is faster than from the cell body. This is functionally significant because it ensures rapid dark adaptation.

Calcium transients in the rhabdomeres of dark- and light-adapted fly photoreceptor cells.

The light response of fly photoreceptor cells is modulated by changes in free Ca(2+) concentration. Fly phototransduction and most processes regulating it take place in or very close to the rhabdomere. We therefore measured the kinetics and the absolute values of the free Ca(2+) concentration in the rhabdomere of fly photoreceptor cells in vivo by making use of the natural optics of the fly's eye. We show that Ca(2+) flowing into the rhabdomere after light stimulation of dark-adapted cells causes fast Ca(2+) transients that reach peak values higher than 200 microM in <20 msec. Approximately 500 msec later, the free Ca(2+) concentration has declined again to approximately 20 microM. The duration of the Ca(2+) transients becomes still shorter, and their size reduced, when the photoreceptor cell is light-adapted. This reduction in duration and size of the Ca(2+) transients is graded with the intensity of the adapting light. The kinetics and absolute values of the free calcium concentration found to occur in the rhabdomere are suitable to mediate the fast feedback signals known to act on the fly phototransduction cascade.

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.

Light dependence of calcium and membrane potential measured in blowfly photoreceptors in vivo.

Light adaptation in insect photoreceptors is caused by an increase in the cytosolic Ca2+ concentration. To better understand this process, we measured the cytosolic Ca2+ concentration in vivo as a function of adapting light intensity in the white-eyed blowfly mutant chalky. We developed a technique to measure the cytosolic Ca2+ concentration under conditions as natural as possible. The calcium indicator dyes Oregon Green 1, 2, or 5N (Molecular Probes, Inc., Eugene, OR) were iontophoretically injected via an intracellular electrode into a photoreceptor cell in the intact eye; the same electrode was also used to measure the membrane potential. The blue-induced green fluorescence of these dyes could be monitored by making use of the optics of the facet lens and the rhabdomere waveguide. The use of the different Ca2+-sensitive dyes that possess different affinities for Ca2+ allowed the quantitative determination of the cytosolic Ca2+ concentration in the steady state. Determining the cytosolic Ca2+ concentration as a function of the adapting light intensity shows that the Ca2+ concentration is regulated in a graded fashion over the whole dynamic range where a photoreceptor cell can respond to light. When a photoreceptor is adapted to bright light, the cytosolic Ca2+ concentration reaches stable values higher than 10 microM. The data are consistent with the hypothesis that the logarithm of the increase in cytosolic Ca2+ concentration is linear with the logarithm of the light intensity. From the estimated values of the cytosolic Ca2+ concentration, we conclude that the Ca2+-buffering capacity is limited. The percentage of the Ca2+ influx that is buffered gradually decreases with increasing Ca2+ concentrations; at cytosolic Ca2+ concentration levels above 10 microM, buffering becomes minimal.