Heteromeric TRPV4/TRPC1 channels mediate calcium-sensing receptor-induced relaxations and nitric oxide production in mesenteric arteries: comparative study using wild-type and TRPC1−/- mice

We have previously provided pharmacological evidence that stimulation of calcium-sensing receptors (CaSR) induces endothelium-dependent relaxations of rabbit mesenteric arteries through activation of heteromeric TRPV4/TRPC1 channels and nitric oxide (NO) production. The present study further investigates the role of heteromeric TRPV4/TRPC1 channels in these CaSR-induced vascular responses by comparing responses in mesenteric arteries from wild-type (WT) and TRPC1-/- mice. In WT mice, stimulation of CaSR induced endothelium-dependent relaxations of pre-contracted tone and NO generation in endothelial cells (ECs), which were inhibited by the TRPV4 channel blocker RN1734 and the TRPC1 blocking antibody T1E3. In addition, TRPV4 and TRPC1 proteins were colocalised at, or close to, the plasma membrane of endothelial cells (ECs) from WT mice. In contrast, in TRPC1-/- mice, CaSR-mediated vasorelaxations and NO generation were greatly reduced, unaffected by T1E3, but blocked by RN1734. In addition, the TRPV4 agonist GSK1016790A (GSK) induced endothelium-dependent vasorelaxations which were blocked by RN1734 and T1E3 in WT mice, but only by RN1734 in TRPC1-/- mice. Moreover, GSK activated cation channel activity with a 6pS conductance in WT ECs but with a 52 pS conductance in TRPC1-/- ECs. These results indicate that stimulation of CaSR activates heteromeric TRPV4/TRPC1 channels and NO production in ECs, which are responsible for endothelium-dependent vasorelaxations. This study also suggests that heteromeric TRPV4-TRPC1 channels may form the predominant TRPV4-containing channels in mouse mesenteric artery ECs. Together, our data further implicates CaSR-induced pathways and heteromeric TRPV4/TRPC1 channels in the regulation of vascular tone

A Cytoplasmic Domain Mutation in ClC-Kb Affects Long-Distance Communication Across the Membrane

BACKGROUND: ClC-Kb and ClC-Ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of ClC-Kb leads to Bartter's Syndrome, a kidney disease. A point mutation in ClC-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 electrophysiological properties of the mutation R538P in both ClC-Kb and ClC-Ka. R538P selectively abolishes extracellular calcium activation of ClC-Kb but not ClC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the ClC-Kb C-terminal domain had either a different oligomeric status or dimerization interface than that of ClC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the ClC-Kb C-terminal domain and used multi-angle light scattering together with a cysteine-crosslinking approach to show that the dimerization interface is conserved between the ClC-Kb and ClC-Ka C-terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface. CONCLUSIONS: The R538P mutation in ClC-Kb, which leads to Bartter's Syndrome, abolishes calcium activation 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 Ca(2+)-activation process for ClC-Kb, and shows that the cytoplasmic domain is important for the channel's electrophysiological properties. In the highly similar ClC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca(2+). This selective outcome indicates that ClC-Ka and ClC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure