pH dependence of extracellular calcium sensing receptor activity determined by a novel technique.
BACKGROUND
Increasing evidence points to the role of the extracellular Calcium Sensing Receptor (CaSR) as a multimodal receptor responding to diverse physiologic stimuli, such as extracellular divalent and polyvalent cations, amino acids, and ionic strength. Within the kidney, these stimuli converge on the CaSR to coordinate systemic calcium and water homeostasis. In this process, the impact of urinary pH changes on the activity of the CaSR has not yet been defined. We therefore performed the present study to analyze the pH sensitivity of the CaSR.


METHODS
To assess the activation state of the CaSR, we developed a new method based on the functional coupling between CaSR activity and gating of calcium sensitive potassium currents mediated by SK4 potassium channels. Two-electrode voltage clamping was used to determine whole cell currents in Xenopus oocytes heterologously expressing rat CaSR and rat SK4 potassium channels.


RESULTS
Coexpression of CaSR and SK4 gave rise to potassium currents that were dependent on CaSR-mediated intracellular calcium release, and thereby corresponded to the activation state of the CaSR. In presence of extracellular calcium, ambient alkalinization above pH 7.5 increased CaSR activity. Evaluation of the CaSR calcium sensitivity at various ambient proton concentrations revealed that this effect was due to a sensitization of the CaSR towards extracellular calcium.


CONCLUSIONS
Coexpression with SK4 potassium channels provides a fast and sensitive approach to evaluate CaSR activity in Xenopus oocytes. As disclosed by this novel technique, CaSR activity is regulated by extracellular pH.
Doroszewicz
Jolanta
J
Department of Pediatrics, Philipps University of Marburg, Marburg, Germany.
Waldegger
Petra
P
Jeck
Nikola
N
Seyberth
Hannsjörg
H
Waldegger
Siegfried
S
D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D015640:Ion Channel Gating; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D024681:Potassium Channels, Calcium-Activated / Q000235:genetics / Q000378:metabolism; D051381:Rats; D044169:Receptors, Calcium-Sensing / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D018076:DNA, Complementary; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C491575:Kcnn4 protein, rat; D024681:Potassium Channels, Calcium-Activated; D044169:Receptors, Calcium-Sensing; D011994:Recombinant Proteins; C473605:extracellular calcium cation-sensing receptor, rat