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

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 Ca2+ ions. Rod CNG channels are directly inhibited by Ca2+-calmodulin (Ca2+/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 Ca2+/CaM-dependent modulation. The molecular basis for Ca2+/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca2+/CaM to a site in the NH2-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH2-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca2+/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 Ca2+/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH2- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca2+/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 NH2- and COOH-terminal regions underlies Ca2+/CaM-dependent inhibition.