PURPOSE: To investigate the role of tyrosine phosphorylation in circadian regulation of cGMP-gated cation channels (CNGCs) of chicken cone photoreceptors. METHODS: Chick retinas were studied on the second day of constant darkness (DD) after several days of entrainment to 12:12 hr light-dark (LD) cycles in vitro. Inside-out patch recordings were made during the subjective day and subjective night to quantify circadian changes in the sensitivity of CNGCs to activation by cGMP after treatment with various tyrosine kinase and tyrosine phosphatase inhibitors. Immunoprecipitation and immunoblot analysis were also used to examine tyrosine phosphorylation of CNGCs and closely associated proteins after separation by conventional and two-dimensional SDS-PAGE. RESULTS: Treatment with tyrosine kinase inhibitors caused a significant decrease in K(1/2) for cGMP activation of CNGCs in patches excised from cones during the subjective day, but had no effect on K(1/2) during the subjective night. Conversely, treatment with a tyrosine phosphatase inhibitor caused a significant increase in the K(1/2) of CNGCs in patches excised during the subjective night but had no effect on channel K(1/2) during the subjective day. Broad spectrum serine-threonine phosphatase inhibitors had no effect. An 85-kDa tyrosine polypeptide that coimmunoprecipitated with CNGC alpha-subunits was detectable at higher levels during the subjective day than during the subjective night. CNGC alpha-subunits were not tyrosine phosphorylated as a function of the time of day. CONCLUSIONS: Circadian control of cone CNGCs appears to entail elevated daytime tyrosine phosphorylation of an approximately 85-kDa auxiliary protein or another subunit of the CNGCs.
PURPOSE: To investigate the role of tyrosine phosphorylation in circadian regulation of cGMP-gated cation channels (CNGCs) of chicken cone photoreceptors. METHODS:Chick retinas were studied on the second day of constant darkness (DD) after several days of entrainment to 12:12 hr light-dark (LD) cycles in vitro. Inside-out patch recordings were made during the subjective day and subjective night to quantify circadian changes in the sensitivity of CNGCs to activation by cGMP after treatment with various tyrosine kinase and tyrosine phosphatase inhibitors. Immunoprecipitation and immunoblot analysis were also used to examine tyrosine phosphorylation of CNGCs and closely associated proteins after separation by conventional and two-dimensional SDS-PAGE. RESULTS: Treatment with tyrosine kinase inhibitors caused a significant decrease in K(1/2) for cGMP activation of CNGCs in patches excised from cones during the subjective day, but had no effect on K(1/2) during the subjective night. Conversely, treatment with a tyrosine phosphatase inhibitor caused a significant increase in the K(1/2) of CNGCs in patches excised during the subjective night but had no effect on channel K(1/2) during the subjective day. Broad spectrum serine-threonine phosphatase inhibitors had no effect. An 85-kDa tyrosine polypeptide that coimmunoprecipitated with CNGC alpha-subunits was detectable at higher levels during the subjective day than during the subjective night. CNGC alpha-subunits were not tyrosine phosphorylated as a function of the time of day. CONCLUSIONS: Circadian control of cone CNGCs appears to entail elevated daytime tyrosine phosphorylation of an approximately 85-kDa auxiliary protein or another subunit of the CNGCs.
Authors: Christophe Paillart; Kai Zhang; Tatiana I Rebrik; Wolfgang Baehr; Juan I Korenbrot Journal: Vis Neurosci Date: 2006 Jan-Feb Impact factor: 3.241