Session: 732 APS Ion Channels, Transporters, and Pumps in Health and Disease Poster Session
(732.15) Effect of a C-terminal Electrostatic Interaction Domain on KCC3 Function
Monday, April 4, 2022
10:15 AM – 12:15 PM
Location: Exhibit/Poster Hall A-B - Pennsylvania Convention Center
Poster Board Number: E260
Alejandrina Flores-Carrillo (INCICh, Universidad La Salle), Diana Mercado-Rodríguez (INCICh, Universidad La Salle), Adriana Mercado (INCICh), Paola de los Heros (UNAM, INCICh)
Presenting Author INCICh, Universidad La Salle CDMX, Mexico City, Mexico
Background
The K:Cl cotransporters (KCCs) are key players to maintain cellular volume, mediate epithelial transport and intracellular chloride concentration, and regulate GABA neurotransmission. Under isotonic conditions, KCCs are normally inhibited due to the phosphorylation of residues located within the cytoplasmic domains. On the contrary, dephosphorylation of those residues in response to hypotonic cell swelling stimulates transport activity. Dysfunction of these proteins and the processes involved in their regulation have been associated with various human diseases by alteration of ionic homeostasis mainly in the kidney and the brain. Based on the cryo-EM structures recently reported of human KCCs, revealing structural determinants for regulation in both N- and C-terminal domains, in the present study we analyzed the effect of a structural domain located at the C- terminal involved in electrostatic interactions. It has been stated that the side chains of residues as Gln (Q), Arg (R) and Lys (K) tend to form hydrogen bonds promoting the stabilization of tertiary and quaternary protein structures. However, whether the phosphorylation status is modified after the electrostatic disruption is unknown.
Methods
Xenopus laevis oocytes and HEK 293 cells were either microinjected or transfected with KCC3a wild type (WT) or C-terminal mutants, Q905A, Q905/K934A (2M), Q905/K934/R937A (3M) and Q905/K934/R937/K938A (4M), key residues involved in electrostatic interactions and located within the C-terminal domain of the cotransporter. The phosphorylation status of KCC3a in response to isotonic and hypotonic conditions was evaluated by immunoblot analysis of three key regulatory phosphorylation sites (pSer96, pThr991 and pThr1048). In parallel, phosphorylation status of SPAK/OSR1 activating regulatory site pSer373/pSer325 was analyzed under the same conditions.
Results
Cell swelling-induced dephosphorylation is not observed on C-terminal electrostatic interaction domain mutants compared to WT KCC3 in X. laevis oocytes and HEK cells. All three regulatory-phosphorylation sites in KCC3a mutants remained phosphorylated under hypotonic conditions where dephosphorylation is required for the protein to become active. These results suggest that the disruption of the hydrogen bonds could disrupt the proper folding of the protein and thus interfere with the regulatory dephosphorylation/phosphorylation signaling pathway which is known to activate the transporter after cell swelling.
Conclusions
Our results show that the four residues in the electrostatic interaction domain of the C-termini are involved in KCC3a response to hypotonic conditions affecting the phosphorylation status of the cotransporter. The side chains of these residues seem to be important in maintaining tertiary conformation given their ability to form hydrogen bonds and thus, the disruption of their interactions affects the cotransporter ability to respond to osmotic and cell volume changes.
