Calmodulation meta-analysis: Predicting calmodulin binding via canonical motif clustering

The calcium-binding protein calmodulin (CaM) directly binds to membrane transport proteins to modulate their function in response to changes in intracellular calcium concentrations. Because CaM recognizes and binds to a wide variety of target sequences, identifying CaM-binding sites is difficult, requiring intensive sequence gazing and extensive biochemical analysis. Here, we describe a straightforward computational script that rapidly identifies canonical CaM-binding motifs within an amino acid sequence. Analysis of the target sequences from high resolution CaM-peptide structures using this script revealed that CaM often binds to sequences that have multiple overlapping canonical CaM-binding motifs. The addition of a positive charge discriminator to this meta-analysis resulted in a tool that identifies potential CaM-binding domains within a given sequence. To allow users to search for CaM-binding motifs within a protein of interest, perform the meta-analysis, and then compare the results to target peptide-CaM structures deposited in the Protein Data Bank, we created a website and online database. The availability of these tools and analyses will facilitate the design of CaM-related studies of ion channels and membrane transport proteins

Counting membrane-embedded KCNE β-subunits in functioning K+ channel complexes

Ion channels are multisubunit proteins responsible for the generation and propagation of action potentials in nerve, skeletal muscle, and heart as well as maintaining salt and water homeostasis in epithelium. The subunit composition and stoichiometry of these membrane protein complexes underlies their physiological function, as different cells pair ion-conducting α-subunits with specific regulatory β-subunits to produce complexes with diverse ion-conducting and gating properties. However, determining the number of α- and β-subunits in functioning ion channel complexes is challenging and often fraught with contradictory results. Here we describe the synthesis of a chemically releasable, irreversible K+ channel inhibitor and its iterative application to tally the number of β-subunits in a KCNQ1/KCNE1 K+ channel complex. Using this inhibitor in electrical recordings, we definitively show that there are two KCNE subunits in a functioning tetrameric K+ channel, breaking the apparent fourfold arrangement of the ion-conducting subunits. This digital determination rules out any measurable contribution from supra, sub, and multiple stoichiometries, providing a uniform structural picture to interpret KCNE β-subunit modulation of voltage-gated K+ channels and the inherited mutations that cause dysfunction. Moreover, the architectural asymmetry of the K+ channel complex affords a unique opportunity to therapeutically target ion channels that coassemble with KCNE β-subunits