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In the mammalian glutamate transporters, countertransported intracellular K+ is essential for relocating the glutamate binding site to the extracellular side of the membrane. This K+-dependent process is believed to be rate limiting for the transport cycle. In contrast, extracellular K+ induces glutamate release upon transporter reversal. Here, we analyzed potential K+ binding sites using molecular dynamics (MD) simulations and site-directed mutagenesis. Two candidate sites were identified by spontaneous K+ binding in MD simulations, one site (K1 site) overlapping with the Na1 Na+ binding site and the K2 site being localized under hairpin loop 2 (HP2). Mutations to conserved amino acid residues in these sites resulted in several transporters that were defective in K+-induced reverse transport and which bound K+ with reduced apparent affinity compared with the wild-type transporter. However, external K+ interaction was abolished in only one mutant transporter EAAC1D454A in the K1 site. Our results, for the first time, directly demonstrate effects of K1-site mutations on K+ binding, in contrast to previous reports on K+ binding sites based on indirect evidence. We propose that K+ binding to the K1 site is responsible for catalyzing the relocation step, whereas binding to the K2 site may have an as-of-yet unidentified regulatory function.

The dynein adaptorDrosophilaBicaudal D (BicD) is auto‐inhibited and activates dynein motility only after cargo is bound, but the underlying mechanism is elusive. In contrast, we show that the full‐length BicD/F684I mutant activates dynein processivity even in the absence of cargo. Our X‐ray structure of the C‐terminal domain of the BicD/F684I mutant reveals a coiled‐coil registry shift; in the N‐terminal region, the two helices of the homodimer are aligned, whereas they are vertically shifted in the wild‐type. One chain is partially disordered and this structural flexibility is confirmed by computations, which reveal that the mutant transitions back and forth between the two registries. We propose that a coiled‐coil registry shift upon cargo‐binding activates BicD for dynein recruitment. Moreover, the human homolog BicD2/F743I exhibits diminished binding of cargo adaptor Nup358, implying that a coiled‐coil registry shift may be a mechanism to modulate cargo selection for BicD2‐dependent transport pathways.