title: Aspartate substitutions establish the concerted action of P-region glutamates in repeats I and III in forming the protonation site of L-type Ca2+ channels.
abstract: Hydrogen ions reduce ion flux through voltage-gated Ca2+ channels by binding to a single protonation site with an unusually high pKa. Recent evidence localizes the protonation site to the same locus that supports high affinity Ca2+ binding and selectivity, a set of four conserved glutamate residues near the external mouth of the pore. Remaining controversy concerns the question of whether the protonation site arises from a single glutamate, Glu-1086 (EIII), or a combination of Glu-1086 and Glu-334 (EI) working in concert. We tested these hypotheses with individual Glu --> Asp substitutions. The Glu --> Asp replacements in repeats I and III stood out in two ways. First, in both EID and EIIID, protonation was destabilized relative to wild type, whereas it was unchanged in EIID and stabilized in EIVD. The changes in affinity were entirely due to alterations in H+ off-rate. Second, the ratio of protonated conductance to deprotonated conductance was significantly closer to unity for EID and EIIID than for wild-type channels or other Asp mutants. Both results support the idea that EI and EIII act together to stabilize a single titratable H+ ion and behave nearly symmetrically in influencing pore conductance. Neutralization of EIII by alanine replacement clearly failed to abolish susceptibility to protonation, indicating that no single glutamate was absolutely required. Taken together, all the evidence supports a model in which multiple carboxylates work in concert to form a single high affinity protonation site.
authors.affiliation: Department of Molecular and Cellular Physiology, Beckman Center, Stanford University Medical Center, Stanford, California 94305, USA. rwtsien@leland.stanford.edu
authors.forename: X H
authors.initials: XH
authors.lastname: Chen
authors.forename: R W
authors.initials: RW
authors.lastname: Tsien
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D015220:Calcium Channels / Q000737:chemistry*; D005971:Glutamates / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D008564:Membrane Potentials; D011817:Rabbits; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
chemical_list: D015220:Calcium Channels; D005971:Glutamates; D011994:Recombinant Proteins; D001224:Aspartic Acid