Tyrosine Phosphorylation of the Inactivating Peptide of the Shaker B Potassium Channel:  A Structural−Functional Correlate

Abstract

A synthetic peptide patterned after the sequence of the inactivating "ball" domain of the Shaker B K + channel restores fast (N-type) inactivation in mutant deletion channels lacking their constitutive ball domains, as well as in K + channels that do not normally inactivate. We now report on the effect of phosphorylation at a single tyrosine in position 8 of the inactivating peptide both on its ability to restore fast channel inactivation in deletion mutant channels and on the conformation adopted by the phosphorylated peptide when challenged by anionic lipid vesicles, a model target mimicking features of the inactivation site in the channel protein. We find that the inactivating peptide phosphorylated at Y8 behaves functionally as well as structurally as the noninactivating mutant carrying the mutation L7E. Moreover, it is observed that the inactivating peptide can be phosphorylated by the Src tyrosine kinase either as a free peptide in solution or when forming part of the membrane-bound protein channel as the constitutive inactivating domain. These findings suggest that tyrosine phosphorylation-dephosphorylation of this inactivating ball domain could be of physiological relevance to rapidly interconvert fast-inactivating channels into delayed rectifiers and vice versa.

N-type Inactivation of the Potassium Channel KcsA by the Shaker B “Ball” Peptide

“…In particular, the ortho (at 7.20 ppm) and meta (at 6.88 ppm) protons of Tyr 8 are among the ShB peptide resonances showing the largest STD effects. This seems consistent with previous findings indicating that Tyr 8 is a critical residue for the ShB peptide to retain its ability to inactivate channels, as well as to adopt a defined, ␤-hairpin conformation upon binding to anionic phospholipid vesicles, a model target mimicking some of the presumed features of the inactivating peptide-binding site on the channel protein (23,55). Interestingly, a conservative Y8F mutation was shown to retain the ability of the wild-type peptide to inactivate potassium channels (16), thus lending support to our findings that the protons at the bulky aromatic ring of Tyr 8 are important in the interaction of the peptide with the target channel during inactivation.…”

Probing the Channel-BoundShakerB Inactivating Peptide by Stereoisomeric Substitution at a Strategic Tyrosine Residue

“…In the absence of inactivating peptides (control conditions, Figure 1), the macroscopic currents elicited by the Shaker B∆6-46 channel inactivate very slowly, with inactivation time constants characteristic of the C-type, slow inactivation process exhibited also by this channel (28). Figure 1 also shows that addition of the wild-type ShB peptide restores very efficiently N-type, rapid channel inactivation in the Shaker B∆6-46 channel, in agreement with previous reports from this and other laboratories (6,30,35). In contrast, addition of the ShB-Y8y peptide instead of the wild-type ShB peptide does not restore rapid channel inactivation, and indeed, the current recordings are very similar to those obtained from the Shaker B∆6-46 channel under control conditions, in the absence of added peptides.…”

Intrinsic Tyrosine Fluorescence as a Tool To Study the Interaction of the Shaker B “Ball” Peptide with Anionic Membranes

“…Nonetheless, the use of anionic lipid vesicles as the inactivating peptide target, mimicking properties of the channel pore, has proven to be an useful and simple model that has allowed to establish a correlation between the adoption of a hydrophobic -hairpin structure by the ShB peptide and its ability to inactivate the channel (10,(12)(13)(14). In this way, modifications at the ShB N-terminal moiety that impede the adoption of the -hairpin in lipid membranes, as is the case of the L7E mutation or the phosphorylation of the Y8 residue, result in noninactivating peptide variants.…”