Molecular determinants of a Ca2+-binding site in the pore of cyclic nucleotide-gated channels: S5/S6 segments control affinity of intrapore glutamates.
Cyclic nucleotide-gated (CNG) channels play an important role in Ca2+ signaling in many cells. CNG channels from various tissues differ profoundly in their Ca2+ permeation properties. Using the voltage-dependent Ca2+ blockage of monovalent current in wild-type channels, chimeric constructs and point mutants, we have identified structural elements that determine the distinctively different interaction of Ca2+ with CNG channels from rod and cone photoreceptors and olfactory neurons. Segments S5 and S6 and the extracellular linkers flanking the pore region are the only structural elements that account for the differences between channels. Ca2+ blockage is strongly modulated by external pH. The different pH dependence of blockage suggests that the pKa of intrapore glutamates and their protonation pattern differ among channels. The results support the hypothesis that the S5-pore-S6 module, by providing a characteristic electrostatic environment, determines the protonation state of pore glutamates and thereby controls Ca2+ affinity and permeation in each channel type.
Seifert
R
R
Forschungszentrum Jülich, Institut für Biologische Informationsverarbeitung, 52425 Jülich, Germany.
Eismann
E
E
Ludwig
J
J
Baumann
A
A
Kaupp
U B
UB
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D054815:Cyclic Nucleotide-Gated Cation Channels; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009712:Nucleotides, Cyclic / Q000378:metabolism; D018034:Olfactory Receptor Neurons / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism; D055672:Static Electricity; D014981:Xenopus
D054815:Cyclic Nucleotide-Gated Cation Channels; D007473:Ion Channels; D009712:Nucleotides, Cyclic; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid