Direct Interaction of a Brain Voltage-Gated K⁺Channel with Syntaxin 1A: Functional Impact on Channel Gating

Abstract

Presynaptic voltage-gated K ϩ (Kv) channels play a physiological role in the regulation of transmitter release by virtue of their ability to shape presynaptic action potentials. However, the possibility of a direct interaction of these channels with the exocytotic apparatus has never been examined. We report the existence of a physical interaction in brain synaptosomes between Kv␣1.1 and Kv␤ subunits with syntaxin 1A, occurring, at least partially, within the context of a macromolecular complex containing syntaxin, synaptotagmin, and SNAP-25. The interaction was altered after stimulation of neurotransmitter release. The interaction with syntaxin was further characterized in Xenopus oocytes by both overexpression and antisense knockdown of syntaxin. Direct physical interaction of syntaxin with the channel protein resulted in an increase in the extent of fast inactivation of the Kv1.1/Kv␤1.1 channel. Syntaxin also affected the channel amplitude in a biphasic manner, depending on its concentration. At low syntaxin concentrations there was a significant increase in amplitudes, with no detectable change in cell-surface channel expression. At higher concentrations, however, the amplitudes decreased, probably because of a concomitant decrease in cell-surface channel expression, consistent with the role of syntaxin in regulation of vesicle trafficking. The observed physical and functional interactions between syntaxin 1A and a Kv channel may play a role in synaptic efficacy and neuronal excitability.

Direct Interaction of Target SNAREs with the Kv2.1 Channel

“…The degenerate phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) (including 5Ј and 3Ј end capping of 2-and 4-phosphorothioates, respectively, and a phosphorothioate at every third internal position to enhance nuclease resistance) was targeted against the following degenerate nucleotide sequence: Asyx, 5Ј-GA(AG)(CU)U(N)CA(UC)GA(CU)AUGUU(CU)AU-GGA(CU)AUG-3Ј (encoding amino acids ELHDMFMDM) corresponds to amino acids 211-219 within the H3 helix of human syntaxin 1A. The ODN is expected to hybridize to syntaxins from human, rodent, bovine, chick, aplysia, leech, and sea urchin homologs as well as to rat syntaxins 3 and 4 (6). The sequence ODN, 5Ј-ATCGTTTGTGAGCGCTTCG-GCATCGGT-3Ј, was used as a non-sense oligomer.…”

“…Immunoprecipitation and Immunoblotting in PC12 Cells-Immunoprecipitation (IP) has been described in detail for synaptosomes (6). In the following, only the changes relevant for PC12 cells are described.…”

“…In Vitro Binding of GST Fusion Proteins with Syx and SNAP-The fusion proteins were synthesized and reacted with Syx as described (6,27). Briefly, purified GST fusion proteins (150 pmol) immobilized on glutathione-Sepharose beads were incubated either with 5 l of the lysate containing 35 S-labeled syntaxin or SNAP (translated on the template of in vitro synthesized mRNAs using a translation rabbit reticulocyte lysate kit (Promega)) or with 200 pmol of recombinant syntaxin peptide prepared from a GST fusion construct (amino acids 1-264) cleaved by thrombin, as described, in 1 ml of phosphate-buffered saline with 0.1% Triton X-100 or 0.5% CHAPS.…”

“…They interact with a wide range of proteins, some of them (such as synaptotagmin) associated with vesicular membranes or with plasma membranes (for example, voltage-gated Ca 2ϩ channels) (1)(2)(3)(4)(5). We suggested previously (6) that SNARE proteins interact with a member of the Kv1 subfamily of the voltage-gated K ϩ (Kv) channels and that these interactions may play a role in synaptic efficacy and neuronal excitability. Our results showed that in brain synaptosomes the presynaptic Kv1.1 channel (7) interacts with some of the protein components of the exocytotic apparatus, including syntaxin 1A, SNAP-25, and synaptotagmin, in a manner that is sensitive to the exocytotic state of the synaptosomes.…”

Calcium/calmodulin-dependent kinase II regulates the interaction between the serotonin transporter and syntaxin 1A

“…Syntaxin 1A has also been shown to directly regulate Ca 2+ channels (Sheng et al, 1994;Bezprozvanny et al, 1995), cystic fibrosis-related Cl − channels (Naren et al, 1997), K + channels (Fili et al, 2001) and epithelial Na + channels (Qi et al, 1999;Saxena et al, 1999). Syntaxin 1A physically interacts with members of the family of plasma membrane neurotransmitter transporters Beckman et al, 1998;Geerlings et al, 2000;Sung et al, 2003), including SERT (Haase et al, 2001) and acts to alter transporter capacity.…”