TY - JOUR
T1 - Identification of a domain on the β-subunit of the rod cGMP-gated cation channel that mediates inhibition by calcium-calmodulin
AU - Grunwald, Maria E.
AU - Yu, Wei Ping
AU - Yu, Hung Hsiang
AU - Yau, King Wai
PY - 1998/4/10
Y1 - 1998/4/10
N2 - The cGMP-gated cation channel mediating phototransduction in retinal rods has recently been shown to be inhibited by calcium-calmodulin, through direct binding of the latter to the β-subunit of the heterotetrameric channel complex. Here, we report the characterization of this inhibition and the identification of a domain crucial for this modulation. Heterologous expression of the α- and β-subunits of the human rod channel in HEK 293 cells produced a cGMP-gated current that was highly sensitive to calcium- calmodulin, with half-maximal inhibition at approximately 4 nM. In biochemical and electrophysiological experiments on deletion mutants of the β-subunit, we have identified a region on its cytoplasmic N terminus that binds calmodulin and is necessary for the calmodulin-mediated inhibition of the channel. However, in gel shift assays and fluorescence emission experiments, peptides derived from this region indicated a low calmodulin affinity, with dissociation constants of approximately 3-10 μM. On the C terminus, a region was also found to bind calmodulin, but it was likewise of low affinity, and its deletion did not abolish the calmodulin-mediated inhibition. We suggest that although the identified region on the N terminus of the β-unit is crucial for the calmodulin effect, other regions are likely to be involved as well. In this respect, the rod channel appears to differ olfactory cyclic nucleotide-gated channel, which is also modulated by calcium-calmodulin.
AB - The cGMP-gated cation channel mediating phototransduction in retinal rods has recently been shown to be inhibited by calcium-calmodulin, through direct binding of the latter to the β-subunit of the heterotetrameric channel complex. Here, we report the characterization of this inhibition and the identification of a domain crucial for this modulation. Heterologous expression of the α- and β-subunits of the human rod channel in HEK 293 cells produced a cGMP-gated current that was highly sensitive to calcium- calmodulin, with half-maximal inhibition at approximately 4 nM. In biochemical and electrophysiological experiments on deletion mutants of the β-subunit, we have identified a region on its cytoplasmic N terminus that binds calmodulin and is necessary for the calmodulin-mediated inhibition of the channel. However, in gel shift assays and fluorescence emission experiments, peptides derived from this region indicated a low calmodulin affinity, with dissociation constants of approximately 3-10 μM. On the C terminus, a region was also found to bind calmodulin, but it was likewise of low affinity, and its deletion did not abolish the calmodulin-mediated inhibition. We suggest that although the identified region on the N terminus of the β-unit is crucial for the calmodulin effect, other regions are likely to be involved as well. In this respect, the rod channel appears to differ olfactory cyclic nucleotide-gated channel, which is also modulated by calcium-calmodulin.
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U2 - 10.1074/jbc.273.15.9148
DO - 10.1074/jbc.273.15.9148
M3 - Article
C2 - 9535905
AN - SCOPUS:0032502770
SN - 0021-9258
VL - 273
SP - 9148
EP - 9157
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 15
ER -