Function and expression of the epithelial Ca2+ channel family: comparison of mammalian ECaC1 and 2

Abstract

1. The epithelial Ca(2+) channel (ECaC) family represents a unique group of Ca(2+)-selective channels that share limited homology to the ligand-gated capsaicin receptors, the osmolarity-sensitive channel OTRPC4, as well as the transient receptor potential family. Southern blot analysis demonstrated that this family is restricted to two members, ECaC1 and ECaC2 (also named CaT1). 2. RT-PCR analysis demonstrated that the two channels are co-expressed in calbindin-D-containing epithelia, including small intestine, pancreas and placenta, whereas kidney and brain only express ECaC1 and stomach solely ECaC2. 3. From an electrophysiological point of view, ECaC1 and ECaC2 are highly similar channels. Differences concern divalent cation permeability, the kinetics of Ca(2+)-dependent inactivation and recovery from inactivation. 4. Ruthenium red is a potent blocker of ECaC activity. Interestingly, ECaC2 has a 100-fold lower affinity for ruthenium red (IC(50) 9 +/- 1 microM) than ECaC1 (IC(50) 121 +/- 13 nM). 5. ECaCs are modulated by intracellular Mg(2+) and ATP. ECaC1 and ECaC2 activity rapidly decay in the absence of intracellular ATP. This effect is further accelerated at higher intracellular Mg(2+) concentrations. 6. In conclusion, ECaC1 and ECaC2 are homologous channels, with an almost identical pore region. They can be discriminated by their sensitivity for ruthenium red and show differences in Ca(2+)-dependent regulation.

The epithelial calcium channels TRPV5 and TRPV6: regulation and implications for disease

“…The inactivation of TRPV6 is characterized by a slow decline after an initial fast inactivation phase, whereas TRPV5 only shows the slow inactivation phase (Nilius et al 2001a. In line with a Ca 2+ -dependent regulation is the observation that chelation of intracellular Ca 2+ with ethylene glycol-bis(b-aminoethyl ether)-N,N,N′,N′-tetra acetic acid (EGTA) or 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acethoxymethylester (BAPTA) resulted in increased channel activity (Bodding and Flockerzi 2004;Hoenderop et al 2001b;Peng et al 1999). Together, these data indicate that intracellular Ca 2+ exerts a negative feedback mechanism on TRPV5 and TRPV6 activity.…”

“…Electrophysiological studies using human embryonic kidney cells (HEK293), heterologously expressing TRPV5 or TRPV6, show that both channels are permeable for monovalent and divalent cations with a high selectivity for Ca 2+ (Gunthorpe et al 2002;Hoenderop et al 2001b;Vennekens et al 2002). The characteristic pore region of TRPV5 and TRPV6 is unique for its high Ca 2+ selectivity, in which a single aspartic residue at position number 542 (D542) is crucial for Ca 2+ permeation Vennekens et al 2001).…”

Molecular Mechanisms of Primary Hypercalciuria

“…The identity of the apical calcium channel in distal tubule remains somewhat controversial; Hoenderop et al (119) did not detect CaT1 RNA in human kidney RNA using RT-PCR but did find abundant ECaC RNA, while Peng et al (120,121) found evidence of CaT1 RNA in human kidney RNA as well as ECaC, localized to distal nephron. Abundance studies using real-time PCR suggest that CaT1 RNA is more abundant than ECaC RNA in kidney (122).…”

GABA uptake-dependent Ca 2+ signaling in developing olfactory bulb astrocytes

“…Application of 600 M 2-APB resulted in Ca 2ϩ oscillations in olfactory bulb astrocytes, suggesting activation of TRP channels. Addition of 10 M ruthenium red, which also acts as a TRPV inhibitor (30), suppressed the generation of Ca 2ϩ oscillations, although an initial Ca 2ϩ transient was elicited frequently upon incubation with 2-APB/ruthenium red (Fig. 4C).…”