CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions

摘要

Cyclic nucleotide-gated (CNG) channels are critical for sensory transduction in retinal photoreceptors and olfactory receptor cells; their activity is modulated by phosphoinositi-des (PIP_n) such as phosphatidylinositol 4,5-bisphosphate (PIP_2) and phosphatidylinositol 3,4,5-trisphosphate (PIP_3). An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a carboxyl (COOH)-terminal leucine zipper domain shown previously to be important for channel assembly and PIP_n regulation. We determined the functional consequences of this mutation using elec-trophysiological recordings of patches excised from cells expressing wild-type and mutant CNG channel subunits. CNGA3-L633P sub-units formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PIP_n inhibition of apparent cGMP affinity for these channels. The impact of L633P on PIP_n sensitivity depended on an intact amino (NH_2) terminal PIP_n regulation module. These observations led us to hypothesize that L633P enhances PIP_n inhibition by altering the coupling between NH_2- and COOH-terminal regions of CNG A3. A recombinant COOH-terminal fragment partially restored normal PIP_n sensitivity to channels with COOH-terminal truncation, but L633P prevented this effect. Furthermore, coimmunoprecipitation of channel fragments, and thermodynamic linkage analysis, also provided evidence for NH_2-COOH interactions. Finally, tandem dimers of CNGA3 subunits that specify the arrangement of subunits containing L633P and other mutations indicated that the putative interdomain interaction occurs between channel subunits (intersubunit) rather than exclusively within the same subunit (intrasubunit). Collectively, these studies support a model in which intersubunit interactions control the sensitivity of cone CNG channels to regulation by phosphoinositides. Aberrant channel regulation may contribute to disease progression in patients with the L633P mutation.