Gating and Stoichiometry of Heteromeric Kainate Receptors

Abstract

Kainate-type ionotropic glutamate receptors (KARs) assemble primarily as heteromeric complexes at glutamatergic synapses. In most cases, KAR-mediated synaptic events exhibit slow and variable deactivation kinetics in contrast to the fast gating properties typically observed with recombinant KARs. It is still not clear which factors contribute to the slowing of KAR responses at synapses, and it remains to be understood the low affinity neurotransmitter, L-Glu, triggers prolonged activations of KARs. Here, we investigated the biophysical and stoichiometric properties of recombinant heteromeric KARs assembled from the GluK2 and GluK5 receptor subunits. To do this, we used a combination of outside-out patch electrophysiology to examine functionality and a fluorescent subunit counting technique to assess heteromerization. As expected, the degree of heteromerization with GluK2/GluK5 subunits in individual patch recordings showed a positive correlation with slow deactivation kinetics and responsiveness to the agonist, AMPA. Interestingly, preliminary data from subunit counting experiments suggest that the stoichiometry of heteromeric KARs is fixed. Furthermore, electrophysiological experiments reveal that GluK2/GluK5 heteromers are insensitive to external anions and cations. Since both anion and cation binding sites line the interface between KARs subunits, our data suggest that the process of heteromer assembly affects functionality by disrupting this region of the mature protein.