Molecular and functional characterization of a murine calcium-activated chloride channel expressed in smooth muscle.
To identify the gene products responsible for the calcium-activated chloride current in smooth muscle, reverse transcription-PCR with degenerate primers was performed on mouse intestine and other organs. A new member of the CLCA gene family was identified, mCLCA4, that is expressed preferentially in organs containing a high percentage of smooth muscle cells, including intestine, stomach, uterus, bladder, and aorta. Reverse transcription-PCR using template RNA prepared from mouse bladder and stomach smooth muscle layers dissected free of mucosa yielded mCLCA4-specific bands. In situ hybridization with an mCLCA4-specific probe confirmed prominent expression in smooth muscle of major vessels of the heart but not cardiac muscle. High expression was also detected in the gastrointestinal tract, in bronchioles, and in aortic and lung endothelial cells. Transient expression of mCLCA4 in 293T cells resulted in the appearance of a prominent calcium-activated chloride current. Whole-cell currents activated by ionomycin or methacholine were anion-selective and showed minimal rectification or voltage-dependent gating. Similar to endogenous currents in smooth muscle cells, methacholine-induced currents were transient, and spontaneous transient inward currents were occasionally observed at resting membrane potentials. These results link calcium-activated chloride channels in smooth muscle with a gene family whose members have been implicated in cystic fibrosis, cancer, and asthma.
Elble
Randolph C
RC
Cancer Biology Laboratories and Departments of Molecular Medicine and Biomedical Sciences, Cornell University College of Veterinary Medicine, Ithaca, New York 14853, USA.
Ji
Guangju
G
Nehrke
Keith
K
DeBiasio
John
J
Kingsley
Paul D
PD
Kotlikoff
Michael I
MI
Pauli
Bendicht U
BU
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D017403:In Situ Hybridization; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D009130:Muscle, Smooth / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D015220:Calcium Channels; D017931:DNA Primers; D002118:Calcium