Divalent cation selectivity in a cyclic nucleotide-gated ion channel.
Divalent metal cation selectivity was studied in guanosine 3',5'-cyclic monophosphate-gated ion channels. Channels from bovine retina were expressed in Xenopus laevis oocytes, and currents were measured using tight-seal patch recording methods. The ability of divalent cations to block Na+ currents was used to determine the occupancy of divalent cations in the ion conduction pore. At positive membrane voltages, where extracellular divalent cations are near equilibrium with their binding site, the occupancy reflects the affinity of the blocking ion. The selectivity sequence based on relative affinity was Ca2+ > Mg2+ = Sr2+ = Ba2+. In addition to its higher affinity, Ca2+ was more permeant and blocked with a weaker voltage dependence. Ca2+ was the only ion that blocked with a high Hill coefficient (n = 2.7), suggesting the presence of multiple binding sites. When Glu 363, located in the pore-forming region, was mutated to Asp, the affinity of all four ions increased and the selectivity sequence became Ca2+ > Sr2+ > Ba2+ > Mg2+. These results show that the channel is highly selective for Ca2+ and that Glu 363 mediates divalent cation selectivity of the channel.
Park
C S
CS
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
MacKinnon
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R
D000595:Amino Acid Sequence; D000818:Animals; D001464:Barium / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology*; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D015640:Ion Channel Gating; D007473:Ion Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D013324:Strontium / Q000494:pharmacology; D014982:Xenopus laevis
D002413:Cations, Divalent; D007473:Ion Channels; D011994:Recombinant Proteins; D001464:Barium; D006152:Cyclic GMP; D008274:Magnesium; D002118:Calcium; D013324:Strontium