Crosslinking the ligand-binding domain dimer interface locks kainate receptors out of the main open state.

Daniels BA, Andrews ED, Aurousseau MR, Accardi MV, Bowie D..

J Physiol. 2013 Aug 15;591(Pt 16):3873-85. PMID: 23713029

 

Objective:   The simplified way of viewing ionotropic glutamate receptors is that they are in their closed conformation until the ligand is bound, at which time they open and allow full ion conductance.  However, recent evidence has demonstrated that a variety of factors, such as the presence of protons or the concentration of ligand at multiple ligand-binding domains (LBDs), may actually lead to a number of permeability states.  In this paper, the ionotropic kainate-type glutamate receptors (KARs)were examined for changes in conductance under conditions of dimer crosslinking through covalent disulfide bonds.

Methods:    tsA201 cells were transfected with mutant/wild type genes encoding the GluK2 subunit.  Outside-out and inside-out recordings were performed on membrane patches to examine GluK2 responses and single-channel events, respectively.

Results:

·         In response to 10mM glutamate, the peak response in a double cysteine mutant in which bonds are known to form was less than 1/10 that of wild type GluK2.  Single mutants had responses either similar to the wild type or completely absent. This may be due to a masking of the LBD or changes in the channel conductance and desensitization.  Analysis of single channel events and computer modeling demonstrates that crosslinking the LBD does not block desensitization by keeping the channel open, but may block ligand binding and block desensitization

·         Analysis of membrane noise, the double mutant was calculated to have a lower conductance than wild type channels.  This was due to a lack of high-conductance open states seen in wild type channels, presumably due to the crosslinking.

·         By summing 3 and 4 exponential components, it was shown that the wild-type channels have shorter shut-states than the mutants do.

Conclusions:

·         Crosslinking LBDs of KARs must alter multiple aspects of channel function

·         Breaking the disulfide bonds doesn’t restore wild-type function, so other effects must also occur

·         Different glutamate receptors may respond differently to LBD crosslinking

 

-DH