Alternative Splicing Governs Cone CNG Channel Sensitivity to Regulation by Phosphoinositides [Neurobiology]

March 27th, 2014 by Dai, G., Sherpa, T., Varnum, M. D.

Precursor mRNA encoding CNGA3 subunits of cone photoreceptor CNG channels undergoes alternative splicing, generating isoforms differing in the N-terminal cytoplasmic region of the protein. In humans, four variants arise from alternative splicing, but the functional significance of these changes has been a persistent mystery. Heterologous expression of the four possible CNGA3 isoforms alone or with CNGB3 subunits did not reveal significant differences in basic channel properties. How-ever, inclusion of optional exon 3, with or without optional exon 5, produced heteromeric CNGA3+CNGB3 channels exhibiting an approximately two-fold greater shift in K1/2,cGMP after PIP2 or PIP3 application compared to channels lacking the sequence encoded by exon 3. We have previously identified two structural features within CNGA3 that support PIPn regulation of cone CNG channels: N- and C-terminal regulatory modules; specific mutations within these regions eliminated PIPn sensitivity of CNGA3+CNGB3 channels. The exon 3 variant enhanced the component of PIPn regulation that depends on the C-terminal region, rather than the nearby N-terminal region, con-sistent with an allosteric effect on PIPn sensitivity due to altered N-C coupling. Alternative splicing of CNGA3 occurs in multiple species, although the exact variants are not conserved across CNGA3 orthologs. Optional exon 3 appears to be unique to humans, even compared to other primates. In parallel, we found that a specific splice variant of canine CNGA3 removes a region of the protein that is necessary for high sensitivity to PIPn. CNGA3 alternative splicing may have evolved in part to tune the interactions between cone CNG channels and membrane-bound phosphoinositides.