Stargazin and \u03b34 slow the channel opening and closing rates of GluA4 AMPA receptors

Vincen D Pierce,Li Niu

doi:10.1038/s41598-019-45870-0

Abstract

As auxiliary subunits, transmembrane AMPA receptor regulatory proteins (TARPs) are known to enhance macroscopic current amplitude and alter kinetic properties of AMPA receptors on slow time scale, such as desensitization rate. Whether TARPs affect the rate of AMPA channel opening and closing, however, remains elusive. Using a laser-pulse photolysis technique, we investigated the effect of γ-2 (stargazin, a type 1a TARP) and γ-4 (a type 1b TARP) on the channel-opening and channel-closing rate constants (i.e., kop and kcl) of GluA4 homomeric channels. We found both TARPs slow the kop and kcl by 4-fold and 3-fold, respectively, without appreciable change of channel-opening probability, as compared with GluA4 channel alone. On the other hand, γ-4 has a stronger effect on slowing the channel desensitization rate than γ-2; yet, γ-2 causes a much more pronounced left shift of the dose-response relationship by increasing its affinity towards glutamate than γ-4. Our study shows that on the faster time scale, the major impact of TARP association with GluA4 is to lengthen the lifetime of the open channel, which is slow to form, to allow a larger charge transfer through the open channel that closes more slowly, without appreciable change of channel opening probability.

Highlights

AMPA receptors are a subtype of glutamate ion channels
Zhang et al.[19] have proposed that the channel-opening probability (Popen) of an AMPA receptor is increased in the presence of transmembrane AMPA receptor regulatory proteins (TARPs), whereas Soto et al.[15] found the Popen is unchanged when γ-2 is complexed with GluA4
In the laser-pulse photolysis measurement, we have shown that the time course of channel opening in the μs time region can be cleanly separated from the channel desensitization reaction that occurs on the ms time scale

Summary

Introduction

AMPA receptors are a subtype of glutamate ion channels. AMPA receptors mediate the majority of the fast neurotransmission in the mammalian central nervous system (CNS), and they are critical for expression of plasticity[1,2]. Whether TAPRs affect kop and/or kcl remains poorly understood This is largely due to the fact that AMPA receptors open their channels upon binding of glutamate in the microsecond (μs) time region but channels become desensitized even in the millisecond (ms) time domain. Potentiation of the current amplitude is thought to be a result of a faster rate of channel opening when TARPs, such as γ-2, are bound to AMPA receptors – this is a hypothesis formed largely from measurements on a slower time scale, i.e. deactivation and desensitization rates[16]. To measure the rate of GluA4 channel opening, we use a laser-pulse photolysis technique, combined with whole-cell current recording By this technique, glutamate is generated photolytically from “caged glutamate” or γ-O-(α-carboxy-2-nitrobenzyl)glutamate with a time constant of ~30 μs[30,31]. Whether potentiation of the macroscopic current amplitude necessarily involves an increase in Popen can be further addressed

Methods

Results

Conclusion