McCabe SL et al. (2004), All-trans-retinal is a closed-state inhibitor o...

XB-ART-3703

J Gen Physiol 2004 May 01;1235:521-31. doi: 10.1085/jgp.200409011.

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All-trans-retinal is a closed-state inhibitor of rod cyclic nucleotide-gated ion channels.

McCabe SL , Pelosi DM , Tetreault M , Miri A , Nguitragool W , Kovithvathanaphong P , Mahajan R , Zimmerman AL .

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Rod vision begins when 11-cis-retinal absorbs a photon and isomerizes to all-trans-retinal (ATR) within the photopigment, rhodopsin. Photoactivated rhodopsin triggers an enzyme cascade that lowers the concentration of cGMP, thereby closing cyclic nucleotide-gated (CNG) ion channels. After isomerization, ATR dissociates from rhodopsin, and after a bright light, this release is expected to produce a large surge of ATR near the CNG channels. Using excised patches from Xenopus oocytes, we recently showed that ATR shuts down cloned rod CNG channels, and that this inhibition occurs in the nanomolar range (aqueous concentration) at near-physiological concentrations of cGMP. Here we further characterize the ATR effect and present mechanistic information. ATR was found to decrease the apparent cGMP affinity, as well as the maximum current at saturating cGMP. When ATR was applied to outside-out patches, inhibition was much slower and less effective than when it was applied to inside-out patches, suggesting that ATR requires access to the intracellular surface of the channel or membrane. The apparent ATR affinity and maximal inhibition of heteromeric (CNGA1/CNGB1) channels was similar to that of homomeric (CNGA1) channels. Single-channel and multichannel data suggest that channel inhibition by ATR is reversible. Inhibition by ATR was not voltage dependent, and the form of its dose-response relation suggested multiple ATR molecules interacting per channel. Modeling of the data obtained with cAMP and cGMP suggests that ATR acts by interfering with the allosteric opening transition of the channel and that it prefers closed, unliganded channels. It remains to be determined whether ATR acts directly on the channel protein or instead alters channel-bilayer interactions.

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Species referenced: Xenopus laevis
Genes referenced: atr camp cnga1 cngb1 rho tbx2

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	Figure 1. . ATR inhibits homomeric (CNGA1) rod channels more potently at low than at saturating cGMP concentrations. Currents were measured from multichannel, inside-out patches of homomeric (CNGA1) rod channels. The raw traces represent families of cGMP-activated currents in response to voltage steps ranging from −100 to +100 mV in 50-mV increments, from a holding potential of 0 mV. Currents measured in the absence of cGMP were subtracted from all traces. (Left) Current families depicting control and inhibition at saturating cGMP (2 mM cGMP) by 300 nM ATR (50.6% inhibition). (Right) Current families showing control and inhibition by 30 nM ATR (67.6% inhibition) at a low cGMP concentration that elicits ∼7.4% of maximal cGMP induced current.
	Figure 2. . The ATR dose–response relations for homomeric (CNGA1) and heteromeric (CNGA1 + CNGB1) rod CNG channels. Cyclic GMP–activated currents were measured at +100 mV before and after ATR addition. Currents were monitored for ∼1 h after ATR addition to ensure that the ATR inhibition had reached steady-state. Averaged data, plotted with SEM (error bars) were fit with the Hill equation (solid or dashed curves): I/I0 = IC50n/(IC50n + [ATR]n), where I is current remaining at a given [ATR], I0 is the current before ATR addition, IC50 is the concentration of ATR required to achieve half maximal inhibition, and n is the Hill (cooperativity) coefficient. Filled circles, homomeric (CNGA1 only) rod channel dose–response relation for inhibition by ATR in saturating (2 mM) cGMP. Data points are averaged from 19 patches; IC50 = 220 nM and n = 1.4. Open triangles, heteromeric (CNGA1 and CNGB1) rod channel dose–response relation for ATR inhibition in saturating (2 mM) cGMP. Data points are from five patches; IC50 = 185 nM and n = 1.8. Open circles, homomeric (CNGA1) rod channel dose–response relation for ATR inhibition at cGMP concentrations evoking ∼7.4% activation. Data are from five patches; IC50 = 20 nM and n = 1.4.
	Figure 3. . cGMP dose–response relation for homomeric (CNGA1) rod channels with and without 400 nM ATR. Steady-state, cGMP-evoked currents were measured at +100 mV from several patches with and without 400 nM ATR. Averaged data were normalized to the maximum current obtained with 2 mM cGMP and no ATR, and fit with an allosteric model (smooth curves; see materials and methods and Fig. 8 for details). Data points are averaged from 2–7 patches, plotted with SEM (error bars). Circles: cGMP dose–response relation without ATR. Model parameters: L = 19.8, KcN = 7,200 M−1. Triangles: cGMP dose–response relation with 400 nM ATR. Model parameters: L = 19.8, KcN = 7,200 M−1, K1 = 9.0 × 1010 M−n, K2 = 4.0 × 1010 M−n, K3 = 2.8 × 1010 M−n, K4 = 2.0 × 109 M−n, R = 400 nM, and n = 1.43.
	Figure 4. . Time course of inhibition by 400 nM ATR in 2 mM cGMP for inside-out and outside-out, multichannel patches containing homomeric (CNGA1) rod channels. (A) Time course of inhibition for this inside-out patch is best fit with a double exponential (τfast = 119 s and τslow = 1,667 s), although patches were often fit with a single exponential. (B) The time course for outside-out patches was so much slower than for inside-out patches that very little inhibition was measured during the lifetime of the patch; for this patch, a single-exponential fit gave τ = 6,748 s, although clearly the current had not reached steady-state by the end of the record.
	Figure 5. . Single-channel recordings in the presence of ATR suggest that the channel can reopen after long-lived closed or inactivated states. Raw current traces in A–C were recorded at a holding potential of +80 mV from an inside-out patch containing one homomeric (CNGA1) channel. Data were filtered at 2 kHz, and the sampling rate was 25 kHz. The lower solid line represents the zero-current level when the channel was closed. The patch was bathed in saturating (2 mM) cGMP (A), and then 200 nM ATR was added to the bath (B and C). (A) A representative portion of a control trace depicting normal channel activity in saturating cGMP; the channel spends the majority of its time open. (B) After adding ATR, the channel activity remained relatively normal for a time. (C) Sometime before the start of this sweep, the channel entered a long-lived closed state, but reopening of the channel (with ATR still in the bath) occurred near the end of the sweep. Of the 3.2 s shown, the channel was in a closed state for 2.5 s; another portion of the record for this patch showed a 6-s closure. These long-lived closed states with ATR have been documented previously (Dean et al., 2002).
	Figure 6. . Current recovered with high cGMP after inhibition by ATR in low cGMP. After applying 100 nM ATR to a bath solution containing low cGMP (activating ∼7.5% of maximal current), the patch current was monitored until the inhibition reached a steady-state (99.5% inhibition). The cGMP concentration in the bath was then raised to 5 mM, with the continued presence of 100 nM ATR in the bath. The increase in [cGMP] opens some channels that were previously unliganded, and also presumably relieves some of the inhibition by ATR, since ATR is a less effective inhibitor in high than in low cGMP (Figs. 1 and 2). After a gap representing the time when cGMP was mixed into the chamber, the current recovery was fit by a single-exponential rise with a time constant of 82 s.
	Figure 7. . Inhibition of homomeric (CNGA1) rod channels by 200 nM ATR in saturating (2 mM) cGMP showed no significant voltage dependence other than that expected with voltage-dependent changes in open probability. For the top panel, an inside-out, multichannel patch was maintained at a holding potential of +50 mV in 200 nM ATR and 2 mM cGMP and monitored to steady-state. A double exponential provided the best fit to the time course of the inhibition, with τfast = 206 s and τslow = 3,779 s. After steady-state was achieved, the holding potential was changed to −50 mV. The time course of the resulting decrease in current (increase in inhibition) was best fit with a single exponential, with τ = 44 s. For the lower panel, the same experiment was performed on another patch, except that the initial membrane potential was −50 mV, and it was then switched to +50 mV. A double exponential provided the best fit to the initial time course of inhibition, with τfast = 77 s and τslow = 769 s. The time course of the increase in current (decrease in inhibition) after switching the voltage to +50 mV was best fit with a single exponential, with τ = 64 s. These changes in ATR inhibition with voltage are consistent with the expected voltage-dependent changes in open probability and the greater ATR inhibition of closed versus open channels.
	Figure 8. . ATR appears to prefer closed, unliganded channels, and to interfere with channel opening. ATR dose–response relations for homomeric (CNGA1) channels activated by cGMP or cAMP. (A) Data were measured as described in Fig. 2 for ATR dose–response curves in saturating (2 mM) cGMP (blue filled circles), saturating (20 mM) cAMP (red squares; giving only 5% of the activation obtained with 2 mM cGMP), and low (∼10–15 μM) cGMP (green open circles; giving ∼7.4% of the activation obtained with 2 mM cGMP). Error bars are SEM values. Averaged data points from 2–7 patches were normalized to Io and fit with the model (B) described above (see materials and methods and results), where K1 = 9.0 × 1010 M−n, K2 = 4.0 × 1010 M−n, K3 = 2.8 × 1010 M−n, K4 = 2.0 × 109 M−n, and n = 1.43. For saturating and low cGMP, L = 19.8 and KcN = 7,200 M−1; for cAMP, L = 0.055 and KcN = 1,144 M−1. The red dashed line represents the prediction of the model for cAMP if ATR binding depends only on the open probability and not on ligand occupancy (i.e., K1 = K2 = K3 = 9 × 1010 M−n).

References [+] :

Ahn, The effect of lipid environment and retinoids on the ATPase activity of ABCR, the photoreceptor ABC transporter responsible for Stargardt macular dystrophy. 2000, Pubmed