Gene Structure and Chromosomal Mapping of Human Epithelial Calcium Channel Dominik Mu¨ ller,* Joost G. J. Hoenderop,* Gerard F. M. Merkx,² Carel H. van Os,* and Rene´ J. M. Bindels* ,1 *Department of Cell Physiology and ²Department of Human Genetics, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Received July 10, 2000 The epithelial Ca 21 channel, ECaC, represents the rate-limiting step of vitamin D 3 -regulated Ca 21 (re)ab- sorption in kidney and intestine, and provides, there- fore, a new candidate gene for Ca 21 -related disorders. To supply the basis for direct mutation analysis, we report here the structure of the human ECaC gene (ECAC1 2 ). It consists of 16 exons spanning 25 kb with introns ranging from 98 to 8500 bp. The 5*-flanking region of ECAC1 contains four putative vitamin D 3 - responsive elements. At positions 292 and 213 tran- scription initiation sites were identified, but the former lacks the canonical TATA or CAAT boxes. ECAC1 was mapped to chromosome 7q35 by fluores- cent in situ hybridization, reassigning a previous ra- diation hybrid mapping to 7q31.1-2. The gene of a re- cently identified rat intestine homologue of ECaC, named Ca 21 transporter 1, was found juxtaposed to the ECaC gene, indicating that both genes are the prod- ucts of evolutionary local gene duplication. © 2000 Academic Press Key Words: calcium absorption; calcium reabsorp- tion; calcitriol; EcaC. The recently identified epithelial Ca 21 channel, ECaC, constitutes the apical Ca 21 influx pathway in 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 )-responsive epithelia present in kidney and small intestine [1– 6]. ECaC exhibits distinctive properties including a con- stitutively activated Ca 21 permeability at physiological membrane potentials, a high selectivity for Ca 21 , an anomalous mole-fraction behavior and hyperpolariza- tion-stimulated and Ca 21 -dependent feedback regula- tion of channel activity [3]. To date, ECaC has been cloned from three different species including rabbit (Accession No. AJ133128), rat (Accession No. AB032019), and human (Accession No. AJ271207). The obtained sequences exhibit an overall homology on amino acid level of about 85% [5]. Recently, the ECaC family has been extended by Peng et al. who identified a Ca 21 transporter (CaT1) from rat small intestine, which is highly homologues to ECaC [7]. These channels represent the first members of a new family of Ca 21 -selective channels involved in active Ca 21 (re)absorption. This transcellular Ca 21 pathway confers the only mechanism by which the organism can influence the Ca 21 uptake in kidney and intestine spe- cifically. ECaC could, therefore, play a crucial role in Ca 21 homeostasis and is a serious candidate gene in disorders associated with alterations of absorption or excretion of Ca 21 [4]. Dysfunctioning of the channel, either as a result of mutations in the gene itself or its regulatory pathways, could ultimately impair the Ca 21 conserving capacity of the body contributing to Ca 21 - associated kidney stone disease and age-related bone disorders. The aim of the present study was to elucidate the gene structure and chromosomal localization of human ECaC to allow mutation analysis in patients suffering from Ca 21 homeostasis-related disorders. MATERIALS AND METHODS Library screening and long range PCR. To determine the genomic organization of ECaC, a PAC clone 209-H-9 was isolated from the RPCI-6 human PAC library by PCR-screening. The approx- imate insert length of this clone was estimated by digestion with different restriction enzymes (BamHI, EcoRI, and HindIII). Clone 209-H-9 was then digested with BamHI and EcoRI and the frag- ments were randomly subcloned into a pBluescript II KS (1/2) vector (Stratagene). These subclones were sequenced by means of standard procedures using Sp6 and T3 promoter primers on a semi- The sequence of the human ECaC gene has been deposited with the EMBL/GenBank Data Libraries under Accession No. AJ401155. 1 To whom correspondence should be addressed at 162 Cell Phys- iology, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Fax: 131-24-3540525. E-mail: reneb@ sci.kun.nl. 2 The HGMW-approved symbol of the human ECaC gene. Biochemical and Biophysical Research Communications 275, 47–52 (2000) doi:10.1006/bbrc.2000.3227, available online at http://www.idealibrary.com on 47 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.