Horrigan DM et al. (2005), Defining the retinoid binding site in the rod c...

XB-ART-1221

J Gen Physiol 2005 Nov 01;1265:453-60. doi: 10.1085/jgp.200509387.

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Defining the retinoid binding site in the rod cyclic nucleotide-gated channel.

Horrigan DM , Tetreault ML , Tsomaia N , Vasileiou C , Borhan B , Mierke DF , Crouch RK , Zimmerman AL .

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Rod vision is initiated when 11-cis-retinal, bound within rhodopsin, absorbs a photon and isomerizes to all-trans-retinal (ATR). This triggers an enzyme cascade that lowers cGMP, thereby closing cyclic nucleotide-gated (CNG) channels. ATR then dissociates from rhodopsin, with bright light releasing millimolar levels of ATR. We have recently shown that ATR is a potent closed-state inhibitor of the rod CNG channel, and that it requires access to the cytosolic face of the channel (McCabe, S.L., D.M. Pelosi, M. Tetreault, A. Miri, W. Nguitragool, P. Kovithvathanaphong, R. Mahajan, and A.L. Zimmerman. 2004. J. Gen. Physiol. 123:521-531). However, the details of the interaction between the channel and ATR have not been resolved. Here, we explore the nature of this interaction by taking advantage of specific retinoids and retinoid analogues, namely, beta-ionone, all-trans-C15 aldehyde, all-trans-C17 aldehyde, all-trans-C22 aldehyde, all-trans-retinol, all-trans-retinoic acid, and all-trans-retinylidene-n-butylamine. These retinoids differ in polyene chain length, chemical functionality, and charge. Results obtained from patch clamp and NMR studies have allowed us to better define the characteristics of the site of retinoid-channel interaction. We propose that the cytoplasmic face of the channel contains a retinoid binding site. This binding site likely contains a hydrophobic region that allows the ionone ring and polyene tail to sit in an optimal position to promote interaction of the terminal functional group with residues approximately 15 A away from the ionone ring. Based on our functional data with retinoids possessing either a positive or a negative charge, we speculate that these amino acid residues may be polar and/or aromatic.

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

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	Figure 1. Structures of retinoids and retinoid analogues. All-trans-retinal (ATR), all-trans-retinol (ATRol), Î²-ionone, all-trans-C15 aldehyde, all-trans-retinoic acid (RA), all-trans-retinylidene-n-butylamine (ATR-NBu), all-trans-C17 aldehyde, and all-trans-C22 aldehyde. (All-trans-C15, C17, and C22 aldehydes are named based on the total number of carbons, including methyl groups, as per Buczylko et al., 1996. Though depicted as charged, the actual charge states of RA and ATR-NBu within the retinoid binding site are not known.)
	Figure 2. Î²-Ionone, and C15 and C17 aldehydes do not significantly inhibit the homomeric rod CNG channel in the presence of saturating cGMP. Currents were measured from multichannel, inside-out patches of homomeric (CNGA1) rod channels at saturating (2 mM) cGMP. The raw traces represent families of cGMP-activated currents in response to voltage steps ranging from â100 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. Black traces represent currents before the addition of the indicated retinoid; red traces represent currents after 1 h (steady state) in the indicated retinoid. Similar results for Î²-ionone, C15, and C17 aldehyde were obtained with 9, 3, and 15 patches, respectively. (A) There was no change in current with 10 Î¼M Î²-ionone. (B) Currents were reduced by 8% in 1 Î¼M C15 aldehyde. (C) Currents were reduced by 11.5% in 1 Î¼M C17 aldehyde.
	Figure 3. C22 aldehyde is a potent inhibitor of the homomeric rod CNG channel. (A) The raw traces represent families of cGMP-activated currents in response to voltage steps ranging from â100 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. Black traces represent currents in saturating cGMP before the addition of C22 aldehyde; red traces represent currents after 1 h (steady state) in 400 nM C22 aldehyde (46% inhibition). (B) The doseâresponse relation for C22 aldehyde inhibition of the rod CNG channel. All points were measured at +100 mV after 1 h (steady state) in the indicated concentration of C22 aldehyde and saturating (2 mM) cGMP. Data were normalized to maximal current in 2 mM cGMP in the absence of C22 aldehyde, with leak current in the absence of cGMP subtracted from each trace. Each point is an average from two to three patches, and the smooth curve is a fit with the Hill equation, where the IC50 = 415 nM, and n = 2.5.
	Figure 4. ATRol is a potent inhibitor of the homomeric rod CNG channel. (A) The raw traces represent families of cGMP-activated currents in response to voltage steps ranging from â100 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. Black traces represent currents in saturating cGMP (2 mM) before the addition of ATRol; red traces represent currents after 1 h (steady state) in 400 nM ATRol (63% inhibition). (B) The doseâresponse relation for ATRol inhibition of the rod CNG channel. All points were measured at +100 mV after 1 h (steady state) in the indicated concentration of ATRol and saturating (2 mM) cGMP. Data were normalized to maximal current in 2 mM cGMP in the absence of ATRol, with leak current in the absence of cGMP subtracted from each. Each point is an average from two to four patches, and the smooth curve is a fit with the Hill equation, where the IC50 = 300 nM, and n = 1.4.
	Figure 5. RA does not inhibit the homomeric rod CNG channel; ATR-NBu does inhibit, but not in a voltage-dependent way. Currents were measured from multichannel, inside-out patches of homomeric (CNGA1) rod channels at saturating (2 mM) cGMP. The raw traces in A and B (top) represent families of cGMP-activated currents in response to voltage steps ranging from â100 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. The traces in B (bottom) represent cGMP-activated currents in response to longer (1.5 s) voltage pulses to +100, +50, â50, or â100 mV from 0 mV after inhibition in 80 nM ATR-NBu reached steady state. The dashed line represents the baseline (i.e., zero current). The holding potential was 0 mV during the application of ATR-NBu. Black traces represent currents in saturating cGMP prior to the addition of RA or ATR-NBu; red or blue traces represent currents after 1 h in RA or ATR-NBu, respectively. (A) 400 nM RA gave no inhibition (red); similar results were seen in two other patches. (B, top) 80 nM ATR-NBu conferred 60% inhibition (blue); (bottom) 1.5-s voltage pulses on the same patch as in the top panel after 1 h in 80 nM ATR-NBu do not show any voltage dependence of ATR-NBu inhibition.

References [+] :

Buczyłko, Mechanisms of opsin activation. 1996, Pubmed