R-type voltage-gated Ca²⁺channels mediate A-type K⁺current regulation of synaptic input in hippocampal dendrites

Abstract

15The transient voltage-gated K + current (IA) mediated by Kv4.2 in CA1 hippocampal pyramidal 16 neurons regulates dendritic excitability, synaptic plasticity, and learning. Here we report that Ca 2+ 17 entry mediated by the voltage-gated Ca 2+ channel subunit Cav2.3 regulates Kv4.2 function in CA1 18 pyramidal neurons through Ca 2+ binding auxiliary subunits known as K + channel interacting 19 proteins (KChIPs). We characterized an interaction between Cav2.3 and Kv4.2 using 20 immunofluorescence colocalization, coimmunoprecipitation, electron microscopy, FRAP, and 21 FRET. We found that Ca 2+ -entry via Cav2.3 increases Kv4.2-mediated whole-cell current due in 22 part to an increase in Kv4.2 surface expression. In hippocampal neurons, pharmacological block 23 of Cav2.3 reduced whole-cell IA. We also found reduced IA in Cav2.3 knockout mouse neurons 24 with a loss of the dendritic IA gradient. Furthermore, the Cav2.3-Kv4.2 complex was found to 25 regulate the size of synaptic currents and spine Ca 2+ transients. These results reveal an 26 intermolecular Cav2.3-Kv4.2 complex impacting synaptic integration in CA1 hippocampal 27 neurons. 28 29 30 31 32 33 34 93 and Kv4.2 that regulates Kv4.2 function by a novel mechanism. 94 95 96 97 98 99 100 101 4 RESULTS 102 103 Cav2.3 and Kv4.2 form an ion channel complex in hippocampal neurons 104The characteristic kinetics of neuronal IA requires expression of KChIP and DPP 105 accessory subunits that had been identified and described nearly two decades ago (27, 28). In 106 an effort to identify novel Kv4.2 protein interactions and modifying enzymes in hippocampal 107 neurons we used tandem affinity purification (TAP) and protein identification with mass 108 spectrometry (MS) (58). As expected, Kv4.2 pulled down Kv4.1, Kv4.2, Kv4.3, and previously 109 described auxiliary subunits (DPPs and KChIPs) confirming the specificity of the assay (Figure 1110 -Figure supplement 1A). In addition to known binding partners, we also identified peptides 111 representing a significant proportion of the Cav2.3 amino acid sequence (Figure 1 -Figure 112 supplement 1B,C). Cav2.3 was the only voltage-gated Ca 2+ channel identified in this screen.113 To confirm the presence of a hippocampal Cav2.3 and Kv4.2 molecular complex we 114 carried out several immunological measurements using hippocampal tissue. Broad neuropil 115 colocalization of endogenous Cav2.3 and Kv4.2 immunofluorescent signal was consistent with 116 enrichment in the dendrite layers of the hippocampus as has been previously reported for each 117 channel separately (Figure 1A) (51, 59). Both Cav2.3 and Kv4.2 are also known to be expressed 118 in dendrites and spines. To evaluate Cav2.3-Kv4.2 colocalization, we transfected cultured 119 hippocampal neurons with Cav2.3-GFP (i), Kv4.2-myc (ii), and mCherry (iii) plasmids (Figure 1B). 120In addition to immunofluorescent colocalization in dendrites, both channels were enriched in 121 spines when compared to the free cytosolic mCherry fluorescent protein (Figure 1C,D). 122Immunofluorescent...