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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 - J. Gen. Physiol. (2005)

Bottom Line: Results obtained from patch clamp and NMR studies have allowed us to better define the characteristics of the site of retinoid-channel interaction.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown Medical School, Providence, RI 02912, USA.

ABSTRACT
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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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 −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. The traces in B (bottom) represent cGMP-activated currents in response to longer (1.5 s) voltage pulses to +100, +50, −50, or −100 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.
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fig5: 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 −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. The traces in B (bottom) represent cGMP-activated currents in response to longer (1.5 s) voltage pulses to +100, +50, −50, or −100 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.

Mentions: We next examined retinoid analogues with terminal functional groups that may carry a negative (all-trans-retinoic acid [RA]) or a positive (all-trans-retinylidene-n-butylamine [ATR-NBu]) charge (see DISCUSSION). Fig. 5 A shows that 400 nM RA did not inhibit the channel. If the site contains aromatic and/or polar residues, then ATR-NBu should be a very effective inhibitor. Fig. 5 B (top) shows a representative family of traces in which 80 nM ATR-NBu conferred 60% inhibition in the presence of saturating (2 mM) cGMP. ATR-NBu was a much more potent inhibitor than any other retinoid tested (see Table I). These results suggest that the retinoid binding site prefers a positively charged over a negatively charged functional group. It is possible that ATR-NBu is acting as a slow voltage-dependent blocker like dequalinium (Rosenbaum et al., 2003, 2004), at a site distinct from the retinoid binding site. However, this does not seem to be the case. Fig. 5 B (bottom) shows a representative set of traces from the same patch as that shown in the top panel, in which each voltage pulse was applied for 1.5 s in the presence of 80 nM ATR-NBu and 2 mM cGMP after steady state was reached. There is no slow increase in current at negative voltages, or decrease in current at positive voltages characteristic of voltage-dependent blockers like dequalinium (Rosenbaum et al., 2004). Thus, ATR-NBu does not appear to be a slow voltage-dependent blocker, but instead most likely inhibits via the same mechanism as the other retinoids tested.


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 - J. Gen. Physiol. (2005)

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 −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. The traces in B (bottom) represent cGMP-activated currents in response to longer (1.5 s) voltage pulses to +100, +50, −50, or −100 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.
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fig5: 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 −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. The traces in B (bottom) represent cGMP-activated currents in response to longer (1.5 s) voltage pulses to +100, +50, −50, or −100 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.
Mentions: We next examined retinoid analogues with terminal functional groups that may carry a negative (all-trans-retinoic acid [RA]) or a positive (all-trans-retinylidene-n-butylamine [ATR-NBu]) charge (see DISCUSSION). Fig. 5 A shows that 400 nM RA did not inhibit the channel. If the site contains aromatic and/or polar residues, then ATR-NBu should be a very effective inhibitor. Fig. 5 B (top) shows a representative family of traces in which 80 nM ATR-NBu conferred 60% inhibition in the presence of saturating (2 mM) cGMP. ATR-NBu was a much more potent inhibitor than any other retinoid tested (see Table I). These results suggest that the retinoid binding site prefers a positively charged over a negatively charged functional group. It is possible that ATR-NBu is acting as a slow voltage-dependent blocker like dequalinium (Rosenbaum et al., 2003, 2004), at a site distinct from the retinoid binding site. However, this does not seem to be the case. Fig. 5 B (bottom) shows a representative set of traces from the same patch as that shown in the top panel, in which each voltage pulse was applied for 1.5 s in the presence of 80 nM ATR-NBu and 2 mM cGMP after steady state was reached. There is no slow increase in current at negative voltages, or decrease in current at positive voltages characteristic of voltage-dependent blockers like dequalinium (Rosenbaum et al., 2004). Thus, ATR-NBu does not appear to be a slow voltage-dependent blocker, but instead most likely inhibits via the same mechanism as the other retinoids tested.

Bottom Line: Results obtained from patch clamp and NMR studies have allowed us to better define the characteristics of the site of retinoid-channel interaction.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown Medical School, Providence, RI 02912, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus