Metabotropic glutamate receptor-mediated suppression of an inward rectifier current is linked via a cGMP cascade

Abstract

Glutamate, the neurotransmitter released by photoreceptors, excites horizontal cells and OFF-type bipolar cells by activating ionotropic receptors. This study investigated an additional action of glutamate in which it modulates a voltage-gated ion channel in horizontal cells. We find that glutamate and APB (2-amino-4-phosphonobutyrate) produce a delayed and moderately prolonged suppression of an inward rectifier current (IRK+). This effect is proposed to occur via an APB-sensitive metabotropic glutamate receptor (mGluR) because common agonists for the ionotropic or APB-insensitive mGluRs are ineffective and the APB-insensitive receptor antagonist alpha-methyl-4-carboxyphenylglycine (MCPG) does not block the actions of glutamate or APB. 8-Br-cGMP, 1-methyl-3-isobutylxanthine (IBMX), and atrial natriuretic peptide (ANP) but not 8-Br-cAMP mimic the suppression of IRK+. The effects of glutamate and APB are blocked by protein kinase inhibitors including Rp-8-pCPT-cGMPS, H-8, and H-7 as well as by ATPgammaS. We hypothesize that the APB receptor suppresses IRK+ via upregulation of cGMP and subsequent activation of a cGMP-dependent protein kinase. This pathway is likely regulated by an ATP-dependent phosphorylation. This is a novel signaling pathway for mGluRs and indicates that at least two distinct APB-activated pathways exist in the retina. Functionally, this APB receptor-mediated action found in horizontal cells would provide a means by which spatially restricted changes of glutamate, produced by local illumination of photoreceptors, could regulate IRK+ and consequently the response properties of these neurons. This would serve to adapt selectively retinal regions stimulated by small regions of the visual world.

Fig. 1.

Characterization of the IR_K⁺ in catfish horizontal cells.A, IR_K⁺ is revealed with voltage ramps of the command potential. Cells were held at −50 mV, stepped to −100 mV, and ramped to +50 mV over a 2 sec period. Ramp durations from 1 to 10 sec produced similar responses. TheI–V relation indicates a current that rectifies near −55 mV, reverses from inward to outward near −60 mV, and increases sharply in magnitude with hyperpolarization (Δ). IR_K⁺ is blocked by external cesium (10 mm) or barium (5 mm) but not by TEA (5 mm). Note that TEA does reduce an outward current at membrane potentials positive to 0 mV, this being an action on the delayed rectifier potassium current. Moreover, TEA shifts the reversal potential slightly positive, suggesting that TEA may be a permeant ion through this inward rectifier channel. B, The cell was held at −50 mV and then stepped to various potentials (−100 to +50 mV) for 1 sec. A 250 msec portion of the current response was averaged (boxed area) and plotted along with the ramp responses (Δ). The current shows a rapid activation and virtually no inactivation. C, IR_K⁺activation (rectification) is a combined function of membrane potential and extracellular K⁺. The extracellular K⁺ concentration ([K⁺]_o) was varied from 0 to 20 mm. With increasing extracellular K⁺, current rectification shifts positive.D, Sodium permeability of the IR_K⁺ channel is shown. Choline substitution for Na⁺ reduces the current magnitude, shifts the reversal potential slightly, but does not change the rectification potential. In 0 mm extracellular Na⁺([Na⁺]_o), IR_K⁺ is still inward at potentials positive to E_K (−82 mV in this example), indicating that choline may also permeate this channel, only less so than Na⁺. All ramps are averages of four successive sweeps.

Fig. 2.

APB and glutamate suppress IR_K⁺. A, 30 μm glutamate, applied for 2.3 min, caused an ∼55% suppression of IR_K⁺ as quantified by the reduction of current at −70 mV. The Glutamate trace was recorded ∼7 min after glutamate removal; the Control trace was recorded just before glutamate application. Glutamate-induced suppression of IR_K⁺persisted well past removal of glutamate, eventually recovering to 95% of control after 10 min in this example (Recovery).B, APB (20 μm) for 2.5 min produced a complete suppression of IR_K⁺. TheControl trace was recorded before APB application; thetrace labeled APB was recorded 3 min after APB removal (see C). IR_K⁺ returned to near control levels after 15 min in this example (Recovery).C, Time course of IR_K⁺suppression is shown. Traces are chart records of the voltage-clamp holding current at −50 mV. In the lowerexample, 30 μm glutamate (solid bar above the trace) produced an ∼60% suppression of IR_K⁺ that recovered after 10 min. Sample expanded ramp responses (#, +) are shown in A. The increased amplitude of the ramp responses during glutamate addition reflects the increased input conductance resulting from ionotropic glutamate receptors. To construct the I–V curves inA, we subtracted the ionotropic component of the current. In the top trace, the cell was exposed to 20 μm APB (2.5 min) as indicated by the solid bar above the trace. During and for a period of time after APB addition, the ramp response magnitude was decreased, indicated by the reduction in amplitude of the vertical response lines.

Fig. 3.

ACPD does not suppress IR_K⁺, nor does MCPG block the action of APB on IR_K⁺. A, 50 μm ACPD was applied to the cell for 2 min. TheControl trace was recorded before ACPD application,ACPD was recorded at the end of the 2 min application, and Recovery was recorded 12 min after ACPD removal.B, After recording of the controlI–V (Control), the cell was bathed in MCPG for 3 min. APB was applied for 2 min starting 30 sec after MCPG. APB and Recovery were recorded just before and 10 min after APB removal. In this figure it is demonstrated that MCPG did not inhibit the actions of APB.

Fig. 4.

8-Br-cGMP but not 8-Br-cAMP reduces IR_K⁺. A, 8-Br-cGMP mimicked the action of APB or glutamate by suppressing IR_K⁺. 8-Br-cGMP (1 mm) in the pipette eliminated IR_K⁺ within 5 min of membrane rupture. B, 8-Br-cAMP (1 mm) failed to affect IR_K⁺ but did influence the calcium current. In this example, even after 25 min, IR_K⁺ is unchanged, whereas the calcium current is effectively eliminated. C, IBMX (100 μm) included in the patch pipette led to a slow, progressive suppression of IR_K⁺. In all of these experiments, the response labeled Control was recorded within 30 sec of membrane rupture; the experimental traces were recorded as indicated on the figure. Alltraces are averages of four successive ramps.D, Time course for the effects of cyclic nucleotides is shown. Each symbol represents one experiment;open symbols are for 8-Br-cGMP (n = 5), and filled symbols are for 8-Br-cAMP (n = 5). Current amplitude represents the percent of control (time, 30 sec) measured at −90 mV for the varioustime points. Complete IR_K⁺ suppression was determined by subsequent application of 1 mm Ba²⁺. Some values exceed 100% limits because the Ba²⁺treatment often shifted the entire I–V relation slightly more negative (see Fig. 1).

Fig. 5.

Kinase inhibitors and ATP analogs block the action of APB and glutamate. A, Glutamate suppression of IR_K⁺ was blocked when the patch pipette contained the kinase inhibitor Rp-8-pPCT-cGMPS (2.5 μm). In this example, a maximal dose of glutamate (50 μm) failed to affect IR_K⁺. Thetrace labeled glutamate was recorded 2 min after glutamate removal. Glutamate produced a normal ionotropic current but failed to reduce IR_K⁺magnitude during or after glutamate application. B, The kinase inhibitor H-8 was included in the patch pipette at a concentration of 20 μm. The I–V curves taken before (Control) and at 1 and 15 min after APB (20 μm) superimposed, indicating that H-8 uncoupled receptor activation from suppression of IR_K⁺. C, ATPγS (2 mm) administered via the patch pipette blocks the action of APB on IR_K⁺. In this example, 20 μm APB causes little if any reduction in IR_K⁺ (compare with Fig.2B,C). Ramps were recorded as described in Figure 2B. Alltraces are averages of four ramps.

Fig. 6.

ANP suppressed IR_K⁺, but SNAP had no effect.A, A 5 min application of ANP (50 nm) produced an ∼70% suppression of IR_K⁺that recovered 22 min after removal. The trace labeled ANP was recorded 2 min after removal of ANP. B, Freshly mixed SNAP was perfused at a concentration of 1 mm. No effect on IR_K⁺ was observed. In both examples, the control trace was recorded before agonist application, and the test traces were recorded after agonist removal. All traces are averages of four successive ramps.

Fig. 7.

Model of glutamate-mediated IR_K⁺ suppression. The simplest scheme consistent with the present data indicates that the APB receptor activates a membrane-bound guanylyl cyclase, thereby increasing intracellular cGMP. cGMP activates a protein kinase that may directly phosphorylate the IR_K⁺ channel; however it is possible that one or more intermediaries is involved. Nevertheless, the result of increased cGMP is a closure of IR_K⁺ channels. The dashed arrow and dashed border around GCsignify the hypothesized pathway by which the APB receptor upregulates the activity of a membrane-bound guanylyl cyclase. The dashed arrows between the kinase and the IR_K⁺ channel signify that the site(s) of action of the kinase has not been established. The dashed box denotes one or more hypothetical intermediate steps, such as activation of a phosphatase, between the cGMP-dependent kinase and IR_K⁺.