FUNCTIONAL AND MOLECULAR-IDENTIFICATION OF A NOVEL CHLORIDE CONDUCTANCE IN CANINE COLONIC SMOOTH-MUSCLE

Swelling-activated or volume-sensitive Cl- currents are found in numer ous cell types and play a variety of roles in their function; however, molecular characterization of the channels is generally lacking. Rece ntly, the molecular entity responsible for swelling-activated Cl- curr ent in cardiac myocytes has been identified as ClC-3. The goal of our study was to determine whether such a channel exists in smooth muscle cells of the canine colon using both molecular biological and electrop hysiological techniques and, if present, to characterize its functiona l and molecular properties. We hypothesized that ClC-3 is present in c olonic smooth muscle and is regulated in a manner similar to the molec ular entity cloned from heart. Indeed, the ClC-3 gene was expressed in colonic myocytes, as demonstrated by reverse transcriptase polymerase chain reaction performed on isolated cells. The current activated by decreasing extracellular osmolarity from 300 to 250 mosM was outwardly rectifying and dependent on the Cl- gradient. Current magnitude incre ased and reversed at more negative potentials when Cl- was replaced by I- or Br-. Tamoxifen ([Z]-1-[p-dimethylaminoethoxy-phenyl]-1,2-diphen yl -1-butene; 10 mu M) and DIDS (100 mu M) inhibited the current, wher eas 25 mu M niflumic acid, 10 mu M nicardipine, and Ca2+ removal had n o effect. Current was inhibited by 1 mM extracellular ATP in a voltage -dependent manner. Cl- current was also regulated by protein kinase C, as phorbol 12,13-dibutyrate (300 nM) decreased Cl- current magnitude, while chelerythrine chloride (30 mu M) activated it under isotonic co nditions. Our findings indicate that a current activated by hypotonic solution is present in colonic myocytes and is likely mediated by ClC- 3. Furthermore, we suggest that the ClC-3 may be an important mechanis m controlling depolarization and contraction of colonic smooth muscle under conditions that impose physical stress on the cells.