A transient receptor potential-like channel mediates synaptic transmission in rod bipolar cells

Abstract

On bipolar cells are connected to photoreceptors via a sign-inverting synapse. At this synapse, glutamate binds to a metabotropic receptor which couples to the closure of a cation-selective transduction channel. The molecular identity of both the receptor and the G protein are known, but the identity of the transduction channel has remained elusive. Here, we show that the transduction channel in mouse rod bipolar cells, a subtype of On bipolar cell, is likely to be a member of the TRP family of channels. To evoke a transduction current, the metabotropic receptor antagonist LY341495 was applied to the dendrites of cells that were bathed in a solution containing the mGluR6 agonists L-AP4 or glutamate. The transduction current was suppressed by ruthenium red and the TRPV1 antagonists capsazepine and SB-366791. Furthermore, focal application of the TRPV1 agonists capsaicin and anandamide evoked a transduction-like current. The capsaicin-evoked and endogenous transduction current displayed prominent outward rectification, a property of the TRPV1 channel. To test the possibility that the transduction channel is TRPV1, we measured rod bipolar cell function in the TRPV1(-/-) mouse. The ERG b-wave, a measure of On bipolar cell function, as well as the transduction current and the response to TRPV1 agonists were normal, arguing against a role for TRPV1. However, ERG measurements from mice lacking TRPM1 receptors, another TRP channel implicated in retinal function, revealed the absence of a b-wave. Our results suggest that a TRP-like channel, possibly TRPM1, is essential for synaptic function in On bipolar cells.

Figure 1.

The rod bipolar cell transduction current is blocked by antagonists of TRPV1. A, Response to 100 μm LY341495 (delivered via fast-flow apparatus) before (left) and after (center) a 5 min application of 10 μm ruthenium red. Right, Response of another cell to a 1 s puff of LY341495 delivered through a puffer pipette alone (top) or during simultaneous application of ruthenium red from a second puffer pipette (middle). Ruthenium red was applied alone for 10 s before obtaining the middle trace. The inhibition of ruthenium red was readily reversed using this approach (bottom). Calibration: 10 pA, 2 s. B, Response to LY341495 before and after a 5 min application of 100 μm 2-APB. C, D, Responses to 1 s puffs of LY341495 (100 μm) before and after 5 min bath application of 20 μm capsazepine (C) and 20 μm SB366791 (D). Responses to LY341495 typically showed partial recovery after removal of antagonists, as shown in the right panel of D. Traces in C and D are from different cells. E, Summary of results. The number of cells for each experiment is indicated above each bar.

Figure 2.

Rod bipolar cells respond to agonists of TRPV1. A, Response to a puff of 10 μm capsaicin in normal solution (left) and in solution containing 20 μm capsazepine (right). B, Response to a puff of 50 μm anandamide in normal bath solution (black traces, left and right) and solution containing 20 μm capsazepine (gray trace, left) or 5 μm AM251, a CB1 receptor antagonist (gray trace, right). Left and right panels are from different cells. C, D, Summary I–V relations for LY341495 (n = 7 cells) and capsaicin (n = 5 cells). Peak currents were normalized to the response at +80 mV for each cell, and the results were pooled. Inset, Responses to LY341495 or capsaicin obtained from representative cells. Voltage steps were from −80 to +80 mV in 20 mV increments. E, F, Plots of normalized conductance for the cells of the transduction channel and the capsaicin-gated channel. Lines are the fits to a Boltzmann function (see Materials and Methods). Plots were obtained from the same sets of cells whose I–V relations are summarized in C and D, using the equation g = I/(V_m − V_rev), where V_rev = 0 mV, to obtain the conductance for each cell.

Figure 3.

Responses to LY31495 and capsaicin occlude each other. A, Responses to 3 s applications of LY341495 and capsaicin, first separately and then together. Also shown in the right panel is the predicted response to simultaneous application based on linear summation. B, Similar to A, except that the duration of the application was 1 s. Note that for subsaturating application of drugs, the predicted response more closely approximates the response to simultaneous application of LY341495 and capsaicin. Same cell as in A. LY, LY31495; Cap, capsaicin. C, Comparison of evoked and predicted responses to simultaneous application of LY31495 and capsaicin as a function of puff duration. Long applications were for a duration of 3 s, whereas brief applications were either 1 s or 0.5 s. Charge was obtained by integration of current during the period of drug application. For both the peak response and the total charge transfer, there was no significant difference between the sizes of the predicted and actual responses to short puffs (p > 0.5; n = 7), but the response to long puffs was significantly smaller than predicted based on summation (p < 0.01; n = 7).

Figure 4.

TRPV1^−/− mice retain the transduction current, TRPV agonist activated currents and ERG b-waves; TRPM^−/− mice lack the ERG b-wave. A, Examples of the responses to LY341495, capsazepine, and anandamide, indicating that both endogenous and exogenous gating of the transduction channel in the TRPV1^−/− is preserved. B, Summary I–V relation for capsaicin in TRPV1^−/− mice (n = 6). Inset, Responses to voltage steps from −80 mV to +80 mV in 20 mV increments. C, Responses to both anandamide and capsaicin were not significantly different in amplitude between wild-type and TRPV1^−/− mice. Ana, Anandamide (WT; n = 13, TRPV1^−/−; n = 4, p > 0.25); Cap, capsaicin (WT; n = 41, TRPV1^−/−; n = 15, p > 0.50). D, Left, Dark-adapted ERGs from a wild-type and a TRPV1^−/− mouse. Flash intensities are (in log cd s/m²) −3.6, −2.4, −1.2, −0.0, and 1.4. Right, Summary of the b-wave and a-wave amplitudes as a function of light intensity in wild-type (n = 8) and TRPV1^−/− (n = 6) mice. E, Left, Dark-adapted ERGs from a wild-type and a TRPM1^−/− mouse. Flash intensities are (in log cd s/m²) −2.6, −1.7, −0.8, 0.1, and 1.0. Right, Summary of the b-wave and a-wave amplitudes as a function of light intensity in wild-type (n = 3) and TRPM1^−/− (n = 4) mice. For TRPM1^−/−, only the a-wave data are plotted, as there was no measurable positive-going response.