Limits...
Dynamics of Ca2+-calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry.

Trudeau MC, Zagotta WN - J. Gen. Physiol. (2004)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Physiology and Biophysics, Box 357290, University of Washington, Seattle, WA 98195-7290, USA.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2233886&req=5

fig2: 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.

Mentions: To determine if the Ca2+/CaM-488 bound to the membrane was indeed the same Ca2+/CaM-488 that was modulating the channels, we compared the time courses of the changes in ionic current and fluorescence intensity recorded simultaneously (Fig. 2). The time course of the current decrease by Ca2+/CaM-488 is similar to that seen previously for Ca2+/CaM (Grunwald et al., 1998; Weitz et al., 1998; Trudeau and Zagotta, 2002b) (Fig. 2 B). The time course of current inhibition and recovery (Fig. 2 B) was mirrored precisely by the time course of fluorescence intensity increase and decrease (Fig. 2 C). Single exponential fits to these data revealed no significant difference between the time constant of current inhibition (τ = 14.7 ± 2.0 s, n = 3) and the time constant for fluorescence increase (τ = 18.5 ± 2.3 s, n = 3). Similarly there was no significant difference between the time constant of current recovery (τ = 68 ± 19 s, n = 3) and the time constant for fluorescence decrease (τ = 51 ± 20 s, n = 3). This similarity between the ionic current and fluorescence intensity time courses suggests that the Ca2+/CaM-488 bound to the membrane was indeed the same Ca2+/CaM-488 that was modulating the channels. The single exponential time course of the fluorescence change is consistent with a single Ca2+/CaM-488 binding to the intact channel, as expected from the 3:1 CNGA1:CNGB1 stoichiometry of the channel. Finally, the similarity between the current and fluorescence time courses indicates that the binding of Ca2+/CaM is the rate-limiting step for channel inhibition.


Dynamics of Ca2+-calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry.

Trudeau MC, Zagotta WN - J. Gen. Physiol. (2004)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233886&req=5

fig2: 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.
Mentions: To determine if the Ca2+/CaM-488 bound to the membrane was indeed the same Ca2+/CaM-488 that was modulating the channels, we compared the time courses of the changes in ionic current and fluorescence intensity recorded simultaneously (Fig. 2). The time course of the current decrease by Ca2+/CaM-488 is similar to that seen previously for Ca2+/CaM (Grunwald et al., 1998; Weitz et al., 1998; Trudeau and Zagotta, 2002b) (Fig. 2 B). The time course of current inhibition and recovery (Fig. 2 B) was mirrored precisely by the time course of fluorescence intensity increase and decrease (Fig. 2 C). Single exponential fits to these data revealed no significant difference between the time constant of current inhibition (τ = 14.7 ± 2.0 s, n = 3) and the time constant for fluorescence increase (τ = 18.5 ± 2.3 s, n = 3). Similarly there was no significant difference between the time constant of current recovery (τ = 68 ± 19 s, n = 3) and the time constant for fluorescence decrease (τ = 51 ± 20 s, n = 3). This similarity between the ionic current and fluorescence intensity time courses suggests that the Ca2+/CaM-488 bound to the membrane was indeed the same Ca2+/CaM-488 that was modulating the channels. The single exponential time course of the fluorescence change is consistent with a single Ca2+/CaM-488 binding to the intact channel, as expected from the 3:1 CNGA1:CNGB1 stoichiometry of the channel. Finally, the similarity between the current and fluorescence time courses indicates that the binding of Ca2+/CaM is the rate-limiting step for channel inhibition.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Physiology and Biophysics, Box 357290, University of Washington, Seattle, WA 98195-7290, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus