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Gucan Dai, Michael Varnum, Cyclic Nucleotide-Gated Ion Channels; Achromatopsia-associated mutation in CNGA3 potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2485.
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© ARVO (1962-2015); The Authors (2016-present)
The ligand sensitivity of retinal cyclic nucleotide-gated (CNG) channels can be adjusted during adaptation and via paracrine or circadian inputs. An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a region of the channel that is important for regulation by phosphoinositides (PIs) such as PIP2 and PIP3. We sought to determine the molecular mechanisms underlying the effect of L633P on channel activity and PI-regulation.
We determined the functional consequences of L633P using electrophysiological recordings of inside-out patches excised from cells expressing cone CNG channel subunits (human CNGA3 and CNGB3). We also used co-immunoprecipitation of channel fragments, thermodynamic linkage analysis, and tandem dimers fixing the arrangement of wild type and/or mutant subunits to examine potential interdomain interactions.
CNGA3-L633P subunits formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PI regulation of apparent cGMP affinity, an effect that depended on an intact N-terminal, rather than C-terminal, PI-regulation module. Recombinant C-terminal fragments partially restored normal PI sensitivity to channels with C-terminal truncation, but L633P prevented this effect. In addition, we detected a physical interaction between N- and C-terminal regions of CNGA3 in vitro, which was disrupted by L633P. These results are consistent with our hypothesis that L633P enhances PI sensitivity by altering the coupling between N- and C-terminal regions of CNGA3. 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). These studies support a model where intersubunit N-C interactions control the PI sensitivity of cone CNG channels.
The achromatopsia associated L633P mutation produces a gain-of-function change in channel gating, increasing the apparent affinity for cGMP. In addition, L633P results in abnormal channel regulation by PIs via disruption of intersubunit coupling. Aberrant PI regulation may contribute to disease progression in patients with the L633P mutation.
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