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Essential role for dlg in synaptic clustering of Shaker K+ channels in vivo.

ABSTRACT: The assemblage of specific ion channels and receptors at synaptic sites is crucial for signaling between pre- and postsynaptic cells. However, the mechanisms by which proteins are targeted to and clustered at synapses are poorly understood. Here we show that the product of the Drosophila discs-large gene, DLG, is colocalized with Shaker K+ channels, which are clustered at glutamatergic synapses at the larval neuromuscular junction. In heterologous cells, DLG can cluster Shaker-type K+ channels, and, in the yeast two-hybrid system, the DLG PDZ1-2 domains bind directly to the C-terminal tail of Shaker proteins. We also demonstrate that DLG-Shaker interactions are required in vivo for Shaker clustering at the neuromuscular junction. Synaptic clustering of Shaker channels is abolished not only by mutations in dlg but also by a mutation in Shaker that deletes its C-terminal DLG binding motif. Analyses of various dlg mutant alleles suggest that channel clustering and synaptic targeting functions depend on distinct DLG domains. These studies demonstrate for the first time that DLG plays an important role in synaptic organization in vivo that correlates with its ability to bind directly to specific membrane proteins of the synapse.

SUBMITTER: Tejedor FJ

PROVIDER: S-EPMC4658234 | biostudies-literature | 1997 Jan

REPOSITORIES: biostudies-literature

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Essential role for dlg in synaptic clustering of Shaker K+ channels in vivo.

Tejedor F J FJ Bokhari A A Rogero O O Gorczyca M M Zhang J J Kim E E Sheng M M Budnik V V

The Journal of neuroscience : the official journal of the Society for Neuroscience 19970101 1

The assemblage of specific ion channels and receptors at synaptic sites is crucial for signaling between pre- and postsynaptic cells. However, the mechanisms by which proteins are targeted to and clustered at synapses are poorly understood. Here we show that the product of the Drosophila discs-large gene, DLG, is colocalized with Shaker K+ channels, which are clustered at glutamatergic synapses at the larval neuromuscular junction. In heterologous cells, DLG can cluster Shaker-type K+ channels, ...[more]

PMID: 8987744

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Project description:K channels mediate the selective passage of K(+) across the plasma membrane by means of intimate interactions with ions at the pore selectivity filter located near the external face. Despite high conservation of the selectivity filter, the K(+) transport properties of different K channels vary widely, with the unitary conductance spanning a range of over two orders of magnitude. Mutation of Pro475, a residue located at the cytoplasmic entrance of the pore of the small-intermediate conductance K channel Shaker (Pro475Asp (P475D) or Pro475Gln (P475Q)), increases Shaker's reported ∼ 20-pS conductance by approximately six- and approximately threefold, respectively, without any detectable effect on its selectivity. These findings suggest that the structural determinants underlying the diversity of K channel conductance are distinct from the selectivity filter, making P475D and P475Q excellent probes to identify key determinants of the K channel unitary conductance. By measuring diffusion-limited unitary outward currents after unilateral addition of 2 M sucrose to the internal solution to increase its viscosity, we estimated a pore internal radius of capture of ∼ 0.82 Å for all three Shaker variants (wild type, P475D, and P475Q). This estimate is consistent with the internal entrance of the Kv1.2/2.1 structure if the effective radius of hydrated K(+) is set to ∼ 4 Å. Unilateral exposure to sucrose allowed us to estimate the internal and external access resistances together with that of the inner pore. We determined that Shaker resistance resides mainly in the inner cavity, whereas only ∼ 8% resides in the selectivity filter. To reduce the inner resistance, we introduced additional aspartate residues into the internal vestibule to favor ion occupancy. No aspartate addition raised the maximum unitary conductance, measured at saturating [K(+)], beyond that of P475D, suggesting an ∼ 200-pS conductance ceiling for Shaker. This value is approximately one third of the maximum conductance of the large conductance K (BK) channel (the K channel of highest conductance), reducing the energy gap between their K(+) transport rates to ∼ 1 kT. Thus, although Shaker's pore sustains ion translocation as the BK channel's does, higher energetic costs of ion stabilization or higher friction with the ion's rigid hydration cage in its narrower aqueous cavity may entail higher resistance.

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Essential role for dlg in synaptic clustering of Shaker K+ channels in vivo.

Publications

Essential role for dlg in synaptic clustering of Shaker K+ channels in vivo.

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