A cytoplasmic domain mutation in CIC-Kb affects long-distance communication across the membrane

Abstract

Background: CIC-Kb and CIC-ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of CIC-Kb leads to Bartters Syndrome, a kidney disease. A point mutation in CIC-Kb, R538P, linked to Bartter's, Syndrome and located in the C-terminal cytoplasmic domain was hypothesized to alter electrophysiological properties due to its proximity to an important membrane embedded helix. Methodology/Principal Findings. Two-electrode voltage clamp experiments were used to examine the psysiological. properties of the mutation R538P in both CIC-Ko and CIC-Ka. R538P selectively abolishes extracellular calcium activation of CIC-Kb but not CIC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the CIC-Kb C-terminal domain had either a different: oligomeric status or dimerization interface than that of CIC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the CIC-Kb C-terminal domain and used multi-angle light: scattering together with a crysteine crosslinking approach to show tha the dimerization interface is conserved between the CIC-Kb and CIC-Ka terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface. Conclusions: The R538P mutation in CIC-Kb, which leads to Bartter's Syndrome, abolishes calciuma ativation of the channel. This suggests that a significant conformational change - ranging from the cytoplasmic side of the protein to the extracellular side of the protein - is involved in the Ca4+ activation process for CIC-kb, and shows that the cytoplasmic domain is important for the channel's e elecrophysiological properties. In the highly similar CIC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca2+. This selective outcome indicates that CIC-Ka and CIC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure. © 2008 Martinez, Maduke.