History The voltage-gated potassium channel Kv1. thus delayed its N-type fast inactivation. Conclusion These data suggest that synaptotagmin I is an interacting protein of Kv1.4 channel and as a negative modulator may play an important role in regulating neuronal excitability and Degrasyn synaptic efficacy. substrate for CaMKII . The phosphorylation of Kv1.4 by CaMKII can modulate the inactivation kinetics of this channel. It is suggested that kinases and phosphatases as well as other signaling and scaffolding proteins may be intimately associated with the ion channel in a regulatory protein complex . Therefore the functional complexity of Kv1.4 will increase as additional associated proteins are found. In the present research synaptotagmin I a Ca2+ sensor playing a key role in the regulation of synaptic vesicle exocytosis  was shown by proteomic strategy to associate with Kv1.4 channel-containing complexes affinity purified from rat hippocampus. Further experiments revealed that there was specific and Ca2+-dependent conversation between synaptotagmin I and Kv1.4 and the conversation was not mediated by other synaptic proteins. Such specific conversation occurred between synaptotagmin I and the N-terminus of Kv1.4 channel and thus the fast N-type inactivation of the channel was delayed. The functional consequence of the conversation between synaptotagmin I and Kv1.4 is that the inactivation kinetics of Kv 1.4 is specifically modulated by synaptotagmin I leading to the decrease in the neuronal excitability. Results Affinity purification and proteomic analysis of native Kv1.4 channel complex Affinity purification and proteomic analysis were employed to identify the components of native Kv1.4 channel complex to find new modulatory factors of the ion channel. After the Kv1.4 channel complex was affinity purified with a Kv1.4-specific antibody (anti-Kv1.4) from plasma membrane-enriched protein fractions prepared from rat hippocampus it was subjected to SDS-PAGE using preimmunization immunoglobulins G (IgGs) as a negative control. The silver-stained protein bands obtained specifically with anti-Kv1.4 but not with preimmunization IgGs (Determine? 1 were selected for protein identification with CapLC-MS/MS. It was shown that this identified proteins included Kv1.1 protein also an A-type Kv channel that had already been demonstrated to interact with Kv1.4  several typical constituents of the synaptic exocytosis machinery (such as syntaxin1B rab3A and synaptotagmin I) and Na+/K+-ATPase as well as cytoskeleton proteins (tubulin and actin) (Table? 1 Synaptotagmin I was identified in the bands at about 47?kDa and 65?kDa (Physique? 1 suggesting that this protein exits in different forms due to the post-translation modification Degrasyn . In view of the functional significance of synaptotagmin  and its abundant copurification with Kv1.4 using anti-Kv1.4 antibody (Figure? 1 the possible conversation between synaptotagmin I and Kv1.4 channel protein was further examined in subsequent investigations. Physique 1 Validation of conversation between synaptotagmin I and Kv1.4 channel. (A) Silver-stained SDS-PAGE of Degrasyn the protein complexes affinity purified from rat hippocampal plasma membrane-enriched fraction either with a Kv1.4-specific antibody (anti-Kv1.4) or a … Table 1 Proteins affinity purified with anti-Kv1.4 from rat hippocampal plasma membranes-enriched protein fraction and identified by CapLC tandem Degrasyn mass spectrometry Rabbit polyclonal to PLD3. The coassembly of synaptotagmin I and Kv1.4 channel complex was confirmed by subsequent reverse copurification from the rat hippocampal plasma membrane-enriched protein preparations with a synaptotagmin I -specific antibody (anti-synaptotagmin I). As illustrated by the western blot in Physique? 1 Kv1.4 Degrasyn was copurified by the anti-synaptotagmin I but not by the pool of preimmunization IgGs used as a control. These results further exhibited that synaptotagmin I can associate with Kv1.4 channel complex. Overlapping expression profile of synaptotagmin I and Kv1.4 in hippocampus Information regarding the distributions of synaptotagmin I and Kv1.4 in neurons is helpful to analyze their conversation. We performed dual labeling experiments to determine whether synaptotagmin I and Kv1.4 are colocalized in hippocampal neurons as would be predicted if the interactions reported here are of physiological relevance. The expression profiles of synaptotagmin I and Kv1.4 in the hippocampus were analyzed.