TRPM3 expression in mouse retina

Transient receptor potential (TRP) channels constitute a large family of cation permeable ion channels that serve crucial functions in sensory systems by transducing environmental changes into cellular voltage and calcium signals. Within the retina, two closely related members of the melastatin TRP family, TRPM1 and TRPM3, are highly expressed. TRPM1 has been shown to be required for the depolarizing response to light of ON-bipolar cells, but the role of TRPM3 in the retina is unknown. Immunohistochemical staining of mouse retina with an antibody directed against the C-terminus of TRPM3 labeled the inner plexiform layer (IPL) and a subset of cells in the ganglion cell layer. Within the IPL, TRPM3 immunofluorescence was markedly stronger in the OFF sublamina than in the ON sublamina. Electroretinogram recordings showed that the scotopic and photopic a- and b-waves of TRPM3(-/-) mice are normal indicating that TRPM3 does not play a major role in visual processing in the outer retina. TRPM3 activity was measured by calcium imaging and patch-clamp recording of immunopurified retinal ganglion cells. Application of the TRPM3 agonist, pregnenolone sulfate (PS), stimulated increases in intracellular calcium in ~40% of cells from wild type and TRPM1(‑/‑) mice, and the PS-stimulated increases in calcium were blocked by co-application of mefenamic acid, a TRPM3 antagonist. No PS-stimulated changes in fluorescence were observed in ganglion cells from TRPM3(-/-) mice. Similarly, PS-stimulated currents that could be blocked by mefenamic acid were recorded from wild type retinal ganglion cells but were absent in ganglion cells from TRPM3-/- mice

Expression of recombinant mouse retinal TRPM3.

<p><b>A, B</b>) Ca²⁺ signals were recorded from CHO-K1 cells transiently transfected with mouse TRPM3 by Fura-2-based ratiometric imaging (n = 8 PS-responsive CHO cells). Signal from control solution (1) and Pregnenolone Sulfate (PS)-activated rises in calcium (2). The PS signal was abolished by pretreatment with mefenamic acid (MA; 3). In (<b>B</b>), black line is average, grey is SEM. C, D) CHO-K1 cells were transiently transfected with mouse mCherry-tagged TRPM3. PS activates an outwardly rectifying current in mCherry—TRPM3 transfected cells identified with mCherry fluorescence. PS-stimulated currents were measured from 11 cells; inward currents at -100 mV were 83 ± 13 pA (SEM) and outward currents were 2175 ± 128 pA at +100 mV. Non-transfected CHO cells show no measurable response to application of PS. The currents were blocked by MA. * in (<b>C</b>) indicate time when the holding potential was ramped from -100 to +100 mV to determine I–V relationship in (<b>D</b>).</p

TRPM3-dependent intracellular calcium increases in mouse retinal ganglion cells.

<p>Ca²⁺ signals were recorded from immunopanned ganglion cells by Fura-2-based ratiometric imaging. Dotted lines indicate where gaps were introduced in some traces to facilitate proper alignment. Greater than 50% of ganglion cells from WT mice responded to Pregnenolone Sulfate (PS)-activated rises in calcium (<b>B</b>) compared to baseline (<b>A</b>); the PS signal was abolished by pretreatment with mefenamic acid (MA; <b>C</b>). In the TRPM3^-/- mice, no cells produced a large response to PS; a few cells, however, gave a small response that was not blocked by MA. At the end of the experiment, cells were depolarized with KCl perfusion to verify viability (<b>D</b>).</p

Pregnenolone sulfate activates a TRPM3-dependent outwardly rectifying current in isolated ganglion cells.

<p>Whole cell currents were recorded from isolated retinal ganglion cells. Holding potential was-60 mV; near vertical current ramps were generated by a voltage ramp from-100 to +100 mV over 250 ms. <b>A</b>) Time course illustrates activation of current by focal extracellular application of PS, and the subsequent inhibition by co-application of MA. <b>B</b>) Current-voltage relation of currents from the cell shown in (<b>A</b>). The PS-activated current is PS-Control and displays strong outward rectification characteristic of TRPM3.</p

TRPM3 is expressed in the IPL and GCL of the mouse retina.

<p>Mouse retina sections were labeled by immunofluorescence for TRPM3. <b>A</b>) Transmitted light DIC image showing the retinal cell layers. <b>B</b>) TRPM3 immunoreactivity is observed in the IPL and GCL of a WT mouse retina. <b>C</b>) No immunoreactivity is detected in a retinal section from a TRPM3^-/- mouse. <b>D</b>) Transverse retinal sections of the inner retina double-labeled for TRPM3 (top, red) and calretinin (middle, green). Visible in the merged image (bottom), the outer, OFF half of the IPL (sublamina a) is more strongly labeled than the inner, ON, sublamina b. <b>E</b>) Imaging of the ganglion cell layer, double-labeled for TRPM3 (top, red) and Brn3a (middle, green). The merged image (bottom) shows that Brn3a-positive ganglion cells express TRPM3. Abbreviations are as follows: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars = 20 μm.</p

Isolation of a full-length TRPM3 cDNA from mouse retina by RT-PCR.

<p>Four segments of TRPM3 were amplified from retina cDNA. The primer’s exon and orientation (sense vs antisense) are indicated. The 5’ and 3’ untranslated regions are shown in light grey, the coding region in dark, and splice variation (see text) in white. Restriction enzyme sites used for sequential cloning of overlap extension products are indicated by diamonds. The location of the <i>Bam</i>HI-<i>Xho</i>I cDNA fragment encoding the antigen used to raise an anti-TRPM3 antiserum is shown.</p

Comparison of the ERGs of TRPM3^-/- and TRPM3^+/+ mice.

<p><b>A</b>) Traces of low pass-filtered scotopic ERGs from TRPM3^+/+ (black) and TRPM3^-/- (red) mice. The flash intensity was-3.3 log(cd-s/m²). Each dashed line is the averaged response from one eye. The thick lines are averages for each genotype. The amplitude (<b>B</b>) and implicit time (<b>C</b>) of the scotopic a- (▲) and b- (■) waves at different light intensities are plotted for TRPM3^+/+ (black) and TRPM3^-/- (red) mice. Data points show the mean with the standard error of the mean. <b>D</b>) Traces of low pass-filtered photopic ERGs from TRPM3^+/+ (black) and TRPM3^-/- (red) mice. The flash intensity was 1.91 log (cd-s/m²) under 100 cd/m² white light background. Each dashed line is the averaged response from one eye. The thick lines are averages for each genotype. The amplitude (<b>E</b>) and implicit time (<b>F</b>) of the photopic a- (▲) and b- (■) waves at different light intensities are plotted for TRPM3^+/+ (black) and TRPM3^-/- (red) mice. Data points show the mean with the standard error of the mean.</p