25 research outputs found
Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters
Potassium-coupled chloride transporters (KCCs) play crucial roles
in regulating cell volume and intracellular chloride concentration.
They are characteristically inhibited under isotonic conditions via
phospho-regulatory sites located within the cytoplasmic termini.
Decreased inhibitory phosphorylation in response to hypotonic cell
swelling stimulates transport activity, and dysfunction of this
regulatory process has been associated with various human
diseases. Here, we present cryo-EM structures of human KCC3b
and KCC1, revealing structural determinants for phosphoregulation in both N- and C-termini. We show that phosphomimetic KCC3b is arrested in an inward-facing state in which
intracellular ion access is blocked by extensive contacts with the
N-terminus. In another mutant with increased isotonic transport
activity, KCC1D19, this interdomain interaction is absent, likely
due to a unique phospho-regulatory site in the KCC1 N-terminus.
Furthermore, we map additional phosphorylation sites as well as a
previously unknown ATP/ADP-binding pocket in the large Cterminal domain and show enhanced thermal stabilization of
other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and
may unlock innovative strategies for drug development
Purification of SLC12A6 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>
Purification of SLC17A5 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>
Purification of MFSD2A from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>
Purification of SLC12A5 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>
Purification of SLC46A3 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>
Purification of SLC12A9 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>
Purification of SLC1A1 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>
Purification of SLC45A4 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>
Purification of SLC25A12 from Expi293FTM Cells
<p>This protocol is used to purify the SLC transporter expressed in Expi293FTM Cells using detergent solubilization followed by a series of different chromatographic methods. Depending on the nature of the expression construct and experimental requirements, the GFP tag can be removed using a proteolytic reaction.</p>