ABSTRACT
To investigate the molecular mechanism of Ca transport in the kidney, we have isolated Ca-permeable channels, rECaC (rat ECaC) and mCaT (mouse CaT1), from rodent kidney, which are recently reported as Ca-transporting proteins. RT-PCR suggested the presence of CaT1 in medullary tubules. It showed 67% homology with rECaC constructing a family. Whole cellular currents in Chinese hamster ovary (CHO) cells were measured by patch clamp. Expression of both proteins exhibited a similar large cation current, a high permeability to Ca, a time-dependent rapid inactivation, and a "run-down." When the pipet contained EGTA, the inactivation and the run-down did not occur. Addition of db-cAMP activated and following rp-cAMPS recovered the mCaT-induced current significantly, whereas no influence was observed in the rECaC-induced one. We conclude that ECaC and CaT are a molecular family of ion channel with similar characteristics, contributing Ca transport in the kidney.
Subject(s)
Calcium Channels/metabolism , Cyclic AMP/analogs & derivatives , Kidney/metabolism , Animals , Bucladesine/pharmacology , CHO Cells , Calcium/metabolism , Calcium Channels/genetics , Calcium Channels/isolation & purification , Cell Membrane Permeability/drug effects , Cloning, Molecular , Cricetinae , Cyclic AMP/pharmacology , Egtazic Acid/pharmacology , In Vitro Techniques , Membrane Potentials/drug effects , Mice , Molecular Sequence Data , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , TRPV Cation Channels , Thionucleotides/pharmacology , TransfectionABSTRACT
We have cloned a cDNA inducing a cation-permeable current (mNSC1) from pancreatic beta-cells, which shows niflumate-sensitive current in Xenopus oocytes. To elucidate the expression in mammalian cells, mNSC1 was expressed in CHO cells. The reversal potential by mNSC1 was shifted toward positive which was significantly reversed by flufenamic acid. Single-channel analysis showed a characteristic of a Ca-activated nonselective cation channel. Therefore, we may conclude that mNSC1 expresses a fenamates-sensitive cation channel, inducing membrane depolarization in a mammalian cell.
Subject(s)
Ion Channels/genetics , Ion Channels/physiology , Animals , CHO Cells , Cloning, Molecular , Cricetinae , DNA, Complementary , Flufenamic Acid/pharmacology , Ion Channels/drug effects , Mammals , Membrane Potentials/drug effects , Oocytes/drug effects , Oocytes/physiology , Patch-Clamp Techniques , Recombinant Proteins/metabolism , Transfection , Xenopus laevisABSTRACT
A homologue of the capsaicin receptor-nonselective cation channel was cloned from the rat kidney to investigate a mechanosensitive channel. We found this channel to be inactivated by membrane stretch and have designated it stretch-inactivated channel (SIC). SIC encodes a 563-amino acid protein with putative six transmembrane segments. The cDNA was expressed in mammalian cells, and electophysiological studies were performed. SIC-induced large cation currents were found to be regulated by cell volume, with currents being stimulated by cell shrinkage and inhibited by cell swelling. Single channel analysis showed a conductance of 250 pS with cation permeability (PCl/PNa < 0.1), and the channel possessed some of the characteristics of a stretch-inactivated channel in that it was permeable to calcium, sensitive to membrane stretch, and blocked by Gd3+. Therefore, we cloned one of the mechanosensitive cation channels of mammals, which is considered to regulate Ca2+ influx in response to mechanical stress on the cell membrane.
