Channel-Opening Kinetic Mechanism of Wild-Type GluK1 Kainate Receptors and a C-Terminal Mutant

Kainate receptor channels are involved in regulating both excitatory and inhibitory neurotransmission.1–3 Abnormal function of kainate receptors, however, has been implicated in some neurological disorders, such as epilepsy.4–6 Kainate receptors have 5 subunits, named as GluK1-5.7 GluK1 (previously known as GluR5),8 the focus of this study, is alternatively spliced at the C-terminus, producing various receptor isoforms.8, 9 Among them, GluK1-2b is 49 amino acids longer than GluK1-2a, the shortest, whereas GluK1-2c, the longest, is generated by an in-frame insertion of nucleotides encoding for an additional 29 amino acids, into the GluK1-2b sequence (Figure S1).9 To date, the understanding of the biophysical properties of kainate receptors at a molecular level emerged largely from studies of GluK2.2 The kinetic properties of GluK1 receptors are not well understood, in part because of their intrinsic low surface expression. GluK1, like the other kainate receptor subunits GluK2 and 3, can form homomeric channels in a heterologous expression system.8, 9 However, GluK1, when expressed in human embryonic kidney (HEK-293) cells, for instance, produces a low current response to even a saturating concentration of agonist.9–12 In a study of GluK1, Ren et al.10 identified an endoplasmic reticulum (ER) retention signal from the intracellular C-terminus of GluK1-2b. The motif consists of a series of positively charged residues or mostly arginines. Among them, Arg896 is critical for its ER retention and low surface expression.10 If this ER retention motif in the wild-type GluK1-2b is disrupted by mutation, the ER exit of the receptor is promoted and the surface delivery is enhanced, resulting in a larger whole-cell current.10 A sizeable whole-cell current response is essential for studying the biophysical properties of a receptor channel. For instance, properties of the closed-channel conformation are studied at a low agonist concentration, which evokes low current response.13 A low current response becomes even lower in the presence of an inhibitor. Furthermore, a sizeable whole-cell current response can serve as a functional output at cell surface for studying trafficking of kainate receptors, which is involved in affecting neurotransmission during synaptic plasticity and neuronal development.1–13 Here we focus on the wild-type GluK1-2b receptor subunit, wild-type GluK1-2a (which does not contain the ER retention signal, unlike the wild-type GluK1-2b) and a mutant GluK1-2b (R896A, R897A, R900A, K901A). GluK1-2a is a commonly used wild-type isoform representative of GluK1 in various studies.9, 11, 12, 14, 15 Mutations of mostly arginines within the ER retention signal located in the C-terminus of GluK1-2b are known to abolish ER retention.10 Therefore, the mutant GluK1-2b receptor lacks the ER-retention signal and is expected to express better than the wild-type GluK1-2b in HEK-293 cells for electrophysiological experiments. The questions we set out to address in this study are: (a) what are the kinetic and equilibrium constants for the opening of the GluK1 channels; (b) does a mutation in the ER-retention signal or the trafficking motif affect these constants; (c) does a GluK1 isoform that lacks the entire ER retention signal (i.e., GluK1-2a) have different channel-opening kinetic constants? To measure these channel-opening constants, we used a laser-pulse photolysis technique with a photolabile precursor of glutamate or caged glutamate,16 which provides ~60 microsecond (μs) time resolution.17–19