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J Gen Physiol
2004 Sep 01;1243:211-23. doi: 10.1085/jgp.200409101.
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Dynamics of Ca2+-calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry.
Trudeau MC
,
Zagotta WN
.
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Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca(2+) ions. Rod CNG channels are directly inhibited by Ca(2+)-calmodulin (Ca(2+)/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca(2+)/CaM-dependent modulation. The molecular basis for Ca(2+)/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca(2+)/CaM to a site in the NH(2)-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH(2)-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca(2+)/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca(2+)/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH(2)- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca(2+)/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH(2)- and COOH-terminal regions underlies Ca(2+)/CaM-dependent inhibition.
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15314069
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Figure 1. . Binding of fluorescent Ca2+/CaM to CNG channels. (A) cGMP-activated ionic currents before (left) and after (center) the addition of Ca2+/CaM-488 and after washout (right). All currents were recorded in response to 50 μM cGMP with a voltage pulse from −60 to 60 mV from a holding voltage of 0 mV. (B) Bright field image of patch pipette and fluorescence images before (left) and after (center) addition of Ca2+/CaM-488, and after washout of Ca2+/CaM-488 (right).
Figure 2. . Ionic current and fluorescence during Ca2+/CaM-dependent modulation. (A) Cartoon image depicting two (of four) subunits in rod CNGA1/CNGB1 channels. (B) Time course of ionic current and (C) fluorescence intensity with the addition (black bar) and washout (open bar) of Ca2+/CaM-488. Red lines show single-exponential fits to the data points.
Figure 3. . FRET between Ca2+/CaM-488 and an eCFP on the CNGB1 subunit. (A) Cartoon depicting eCFP-CNGB1 in which an eCFP molecule is genetically attached to the NH2-terminal region of CNGB1, adjacent to the Ca2+/CaM-binding site. (B) Ionic currents and (C) eCFP fluorescence intensities measured at 480 nm with the addition (black bar) and washout (open bar) of Ca2+/CaM-488. A decrease in eCFP intensity indicates FRET and close proximity with Ca2+/CaM-488. (D) Depiction of CNGA1/eCFPΔCaM-CNGB1 channels in which the NH2-terminal CaM-binding site of CNGB1 was deleted. (E) Ionic currents and (F) eCFP intensities in channels lacking a Ca2+/CaM-binding site.
Figure 4. . Proximity of the NH2-terminal region of CNGB1 and the COOH-terminal region of CNGA1. (A) Spectral method for determining FRET and Ratio A. Emission spectra from whole oocytes were taken from CNGA1-eYFP/eCFP-CNGB1 channels with eCFP attached to the NH2-terminal region of CNGB1 and eYFP attached to the COOH-terminal region of CNGA1 (as in the cartoon) with excitation by a 458-nm laser (red line). The eCFP-only spectra (dotted, cyan line) was determined in a separate experiment with channels that contained only eCFP. The extracted F458 spectra (green line) is the red minus the cyan trace. F488 (black line) is the eYFP emission with excitation by a 488-nm laser. Ratio A was calculated as the F458 spectra normalized by the F488 spectra. (B) Determination of Ratio A0. In a control experiment with CNG channels containing only eYFP (cartoon) we determined the emission spectra from eYFP with excitation at 458 nm, F458 (red trace), and the emission spectra with excitation at 488 nm, F488 (black trace). Ratio A0 was calculated as the F458 spectra normalized by the F488 spectra in channels containing only eYFP.
Figure 5. . Reduction of FRET by deletion of a Ca2+/CaM-binding site. Bar graph of Ratio A–Ratio A0, which is proportional to FRET, for the indicated eCFP- and eYFP-containing CNG channels. A 29% reduction in FRET was found for channels lacking the Ca2+/CaM-binding site in the NH2-terminal region of CNGB1.
Figure 6. . Ca2+/CaM-dependent rearrangements in fluorescent channels. (A) Depiction of CNGA1-eYFP/eCFP-CNGB1 channel. (B) Time course of change in ionic current and (C) fluorescence ratio (F530/F480) with the addition (black bar) and washout (open bar) of Ca2+/CaM. (D) Depiction of CNGA1-eYFP/eCFPΔCaM-CNGB1 channels with a deletion of the NH2-terminal Ca2+/CaM-binding site in CNGB1. The time course of the (E) ionic current and (F) fluorescence ratio in channels lacking the NH2-terminal binding site.
Figure 7. . Molecular mechanism of Ca2+/CaM-dependent inhibition of CNGA1/CNGB1 channels. (A) Depiction of an intersubunit interaction between the NH2-terminal region of CNGB1 subunits and the distal COOH-terminal region of CNGA1 subunits. (B) Ca2+/CaM binding to the NH2-terminal Ca2+/CaM-binding site in CNGB1 results in a rearrangement or separation between the NH2-terminal region of CNGB1 and the COOH-terminal region of CNGA1.
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