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

Abstract

GluK1 is a kainate receptor subunit in the ionotropic glutamate receptor family and can form functional channels when expressed, for instance, in HEK-293 cells. However, the channel-opening mechanism of GluK1 is poorly understood. One major challenge to studying the GluK1 channel is its apparent low surface expression, which results in a low whole-cell current response even to a saturating concentration of agonist. The low surface expression is thought to be contributed by an endoplasmic reticulum (ER) retention signal sequence. When this sequence motif is present as in the wild-type GluK1-2b C-terminus, the receptor is significantly retained in the ER. Conversely, when this sequence is lacking, as in wild-type GluK1-2a (i.e., a different alternatively spliced isoform at the C-terminus) and in a GluK1-2b mutant (i.e., R896A, R897A, R900A and K901A) that disrupts the ER retention signal, there is higher surface expression and greater whole-cell current response. Here we characterize the channel-opening kinetic mechanism for these three GluK1 receptors expressed in HEK-293 cells by using a laser-pulse photolysis technique. Our results show that the wild-type GluK1-2a, wild-type GluK1-2b and the mutant GluK1-2b have identical channel-opening and channel-closing rate constants. These results indicate that the C-terminal ER retention signal sequence, which affects receptor trafficking/expression, does not affect channel-gating properties. Furthermore, as compared with the GluK2 kainate receptor, the GluK1 channel is faster to open, close, and desensitize by at least two-fold, yet the EC50 value of GluK1 is similar to that of GluK2.

Contributions of different kainate receptor subunits to the properties of recombinant homomeric and heteromeric receptors

“…We also found that GluK1 homomers were slightly more sensitive to glutamate, with an average EC 50 of 99.2 ± 7.4 μM (n=5) compared to 208.3 ± 28.1 μM (n=5) for the GluK2 homomers (Fig 2), although this difference was not significant (p>0.5). These EC 50 values are in the general range (200 μM - 800 μM) of those reported by others using electrophysiological techniques to measure current responses in the whole-cell or outside-out patch configurations (Raymond et al, 1993; Heckman et al, 1996; Paternain et al, 1998, Barberis et al, 2008), although relatively few studies have examined the glutamate sensitivity of GluK1 homomeric receptors in the absence of modulators like concanavalin A (Sommer et al, 1992; Han et al, 2012). While the whole-cell configuration does not permit accurate quantification of fast kinetic properties like desensitization, the appearance of the GluK2 receptor currents is consistent with several previous studies, and that of the GluK1 homomers is most similar to the responses most commonly observed from these receptors and designated S-type by other investigators (Swanson and Heinemann, 1998).…”

Functional Roles of the Edited Isoform of GluA2 in GluA2-Containing AMPA Receptor Channels

“…From a series of k obs as a function of the concentration of photolytically released glutamate, we estimated, by regression analysis, k op and k cl using eq 3 (Figures 6B and 6C). 25,26,31,[34][35][36]38 For the data analysis, we took the following steps to constrain some parameters to help regression reach a sensible output. To illustrate this, we use the data of the GluA2Q/2R channel as an example.…”

“…The whole-cell current amplitudes of the released glutamate were compared to the amplitudes of the free glutamate, with reference to the dose− response relation. 35,36 In conjunction with the laser-pulse photolysis technique, a fast solution flow technique with a rise time of 1 ms (90% current response) was used to deliver free glutamate or the caged glutamate and to measure the whole-cell current response for the dose−response relation, desensitization rate, and I−V relationship. 35,36 Data Analysis for Whole-Cell Current Traces from Flow Measurements.…”

“…35,36 In conjunction with the laser-pulse photolysis technique, a fast solution flow technique with a rise time of 1 ms (90% current response) was used to deliver free glutamate or the caged glutamate and to measure the whole-cell current response for the dose−response relation, desensitization rate, and I−V relationship. 35,36 Data Analysis for Whole-Cell Current Traces from Flow Measurements. The analysis of the glutamate-induced AMPA receptor current was based on a general, minimal mechanism of channel opening as shown below: On the basis of this general, minimal mechanism of channel opening, eq 1 was derived…”

R/G editing in GluA2Rflop modulates the functional difference between GluA1 flip and flop variants in GluA1/2R heteromeric channels

“…Here we have designed a comprehensive study that included 20 homomeric and heteromeric AMPA receptors that combine R/G editing isoforms with Q/R editing and flip/flop RNA splicing variants. Using a laser-pulse photolysis technique that provided ~60 µs time resolution 3 , 10 , 56 , 66 , 67 , we have systematically investigated the effect of R/G editing on the channel-opening rate process.…”