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Proc Natl Acad Sci U S A
2002 Jun 11;9912:8424-9. doi: 10.1073/pnas.122015999.
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Mechanism of calcium/calmodulin inhibition of rod cyclic nucleotide-gated channels.
Trudeau MC
,
Zagotta WN
.
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Rod cyclic nucleotide-gated (CNG) channels are heterotetramers comprised of both CNGA1 and CNGB1 subunits. Calcium/calmodulin (Ca(2+)/CaM) binds to a site in the N-terminal region of CNGB1 subunits and inhibits the opening conformational change in CNGA1/CNGB1 channels. Here, we show that polypeptides derived from an N-terminal region of CNGB1 form a specific interaction with polypeptides derived from a C-terminal region of CNGA1 that is distal to the cyclic nucleotide-binding domain. Deletion of the Ca(2+)/CaM-binding site from the N-terminal region of CNGB1 eliminated both Ca(2+)/CaM modulation of the channel and the intersubunit interaction. Furthermore, the interaction was disrupted by the presence of Ca(2+)/CaM. These results suggest that Ca(2+)/CaM-dependent inhibition of rod channels is caused by the direct binding of Ca(2+)/CaM to a site in the N-terminal region in CNGB1, which disrupts the interaction between this region and a distal C-terminal region of CNGA1. The mechanism underlying Ca(2+)/CaM modulation of rod channels is distinct from that in olfactory (CNGA2) CNG channels.
Figure 1 Localization of a region in rod CNG channels necessary for inhibition by Ca2+/CaM. (A) (Left) Cartoon image depicting coexpression of CNGA1 and CNGB1 subunits. (Center) cGMP-activated currents from wild-type CNGA1/CNGB1 channels in the absence (a and c) and presence (b) of Ca2+/CaM. (Right) Time course of current inhibition by Ca2+/CaM. Arrows labeled a–c indicate points that correspond to the current traces (Center). (B) cGMP-activated currents from CNGA1/CNGB1δ2–676 channels and time course of inhibition in the presence of Ca2+/CaM. (C) Lack of effect of Ca2+/CaM on CNGA1/CNGB1δ2–701 channels. (D) Lack of effect of Ca2+/CaM on CNGA1/CNGB1δCaM channels. Several CNG channel domains are labeled in A. GARP, glutamic acid-rich protein.
Figure 2 Functional properties of CNG channels. (A) cGMP dose–response relationships from a representative patch for CNGA1 (â– ), CNGA1/CNGB1 (â–¡), CNGA1/CNGB1δ2–676 (â—), CNGA1/CNGB1δ2–701 (â—‹), and CNGA1/CNGB1δCaM (â–´). Currents were measured at 60 mV, normalized to the maximum value in saturating cGMP (2.5 mM), and superimposed with the Hill equation, I/Imax = 1/(1+ (K1/2/[cGMP])n), with K1/2 = 55 μM and n = 2.5. (B) Fractional activation of CNG channels by cAMP, a partial agonist. Each channel construct is depicted by the same symbol as in A, except for Ca2+/CaM with CNGA1/CNGB1 (bowties). Values were determined by normalizing the current at 60 mV in saturating (16 mM) cAMP (or saturating cAMP with Ca2+/CaM) to that in saturating (2.5 mM) cGMP. Error bars represent the SEM; those not visible are within the symbols. n is ≥ 3 for each point.
Figure 3 Difference in molecular determinants for interdomain interactions in CNGA1/CNGB1 channels versus CNGA2 channels. (A) Cartoon showing bait proteins derived from CNGA2 (gray) and CNGB1 (red) and fish proteins derived from CNGA1 (blue) located directly beneath the corresponding region of a generic CNG subunit. The exact amino acid composition of the fusion proteins are given by the numbers within each associated name. (B) Western blot of biochemical pull-down interaction using CNGB1#677–764 as bait and CNGA2#1–138 as fish. (C) Western blot using CNGA2-derived protein as bait and the same fish proteins as in B.
Figure 4 Requirement of N-terminal Ca2+/CaM binding site in CNGB1 for binding both Ca2+/CaM and a C-terminal domain of CNGA1. (A) Cartoon showing bait and fish fusion proteins and their position relative to a generic CNG channel subunit. (B) Gel showing results of biochemical pull-down interaction assay between bait proteins CNGB1#677–764 and CNGB1#702–764 and fluorescently labeled CaM in the presence of Ca2+ or EDTA. (C) Western blot showing results of pull-down assays with bait proteins CNGB1#677–764 and CNGB1#702–764 and the fish protein CNGA1#497–693.
Figure 5 Disruption of the interaction between CNGA1 and CNGB1 by Ca2+/CaM. (A) Bait proteins derived from CNGB1 (red) and fish protein derived from CNGA1 (blue). (B) Western blot of pull-down experiments with the bait protein CNGB1#677–764 and the fish protein CNGA1#609–693, in the presence of EDTA, Ca2+, or CaM. (C) An intersubunit interaction between the distal C-terminal domain of CNGA1 and an N-terminal domain of CNGB1 and prevention of the interaction by Ca2+/CaM.
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