Cytoplasmic dynein is the multisubunit motor protein for retrograde movement of diverse cargoes to microtubule minus ends. Here, we investigate the function of dynein variants, defined by different intermediate chain (IC) isoforms, by expressing fluorescent ICs in neuronal cells. Green fluorescent protein (GFP)–IC incorporates into functional dynein complexes that copurify with membranous organelles. In living PC12 cell neurites, GFP–dynein puncta travel in both the anterograde and retrograde directions. In cultured hippocampal neurons, neurotrophin receptor tyrosine kinase B (TrkB) signaling endosomes are transported by cytoplasmic dynein containing the neuron-specific IC-1B isoform and not by dynein containing the ubiquitous IC-2C isoform. Similarly, organelles containing TrkB isolated from brain by immunoaffinity purification also contain dynein with IC-1 but not IC-2 isoforms. These data demonstrate that the IC isoforms define dynein populations that are selectively recruited to transport distinct cargoes.
Researchers studied the mechanism by which the anticonvulsant Retigabine enhances the activity of K+ channels and found that the drug stabilizes on open state at resting membrane potentials.
The atrial G protein-regulated inwardly rectifying K+ (GIRK1 and GIRK4) heterotetrameric channels underlie the acetylcholine-induced K+ current responsible for vagal inhibition of heart rate and are activated by the G protein βγ subunits (Gβγ). We used a multistage protein-protein docking approach with data from published structures of GIRK1 and Gβγ to generate an experimentally testable interaction model of Gβγ docked onto the cytosolic domains of the GIRK1 homotetramer. The model suggested a mechanism by which Gβγ promotes the open state of a specific cytosolic gate in the channel, the G-loop gate. The predicted structure showed that the Gβ subunit interacts with the channel near the site of action for ethanol and stabilizes an intersubunit cleft formed by two loops (LM and DE) of adjacent channel subunits. Using a heterologous expression system, we disrupted the predicted GIRK1- and Gβγ-interacting residues by mutation of one protein and then rescued the regulatory activity by mutating reciprocal residues in the other protein. Disulfide crosslinking of channels and Gβγ subunits with cysteine mutations at the predicted interacting residues yielded activated channels. The mechanism of Gβγ-induced activation of GIRK4 was distinct from GIRK1 homotetramers. However, GIRK1-GIRK4 heteroterameric channels activated by Gβγ displayed responses indicating that the GIRK1 subunit dominated the response pattern. This work demonstrated that combining computational with experimental approaches is an effective method for elucidating interactions within protein complexes that otherwise might be challenging to decipher.
Inwardly rectifying potassium channels enforce tight control of resting membrane potential in excitable cells. The Kir3.2 channel, a member of the Kir3 subfamily of G-protein-activated potassium channels (GIRKs), plays several roles in the nervous system, including key responsibility in the GABA B pathway of inhibition, in pain perception pathways via opioid receptors, and is also involved in alcoholism. PKC phosphorylation acts on the channel to reduce activity, yet the mechanism is incompletely understood. Using the heterologous Xenopus oocyte system combined with molecular dynamics simulations, we show that PKC modulation of channel activity is dependent on Ser-196 in Kir3.2 such that, when this site is phosphorylated, the channel is less sensitive to PKC inhibition. This reduced inhibition is dependent on an interaction between phospho-Ser (
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