The rSK3 gene encodes a small conductance Ca2+-activated K+ channel that is transcriptionally regulated by estrogen. To examine determinants of rSK3 gene expression, the CAP site was defined and the promoter was identified. A 33 bp sequence adjacent to the promoter was shown to act as an enhancer in L6 cells that express SK3 and estrogen receptor alpha (ERalpha). The 33bp enhancer was unable to stimulate transcription in Cos7 cells that do not express SK3 or ERalpha. However when cotransfected with ERalpha and stimulated with 17beta-estradiol (E2) the enhancer was activated. Interestingly, expression of ERalpha in Cos7 cells and E2 treatment was sufficient to induce expression of the endogenous SK3 gene. Only Sp1 and Sp3 specifically bound to the enhancer from Cos7 and L6 nuclear extracts, however, ERalpha increased Sp1s affinity for the enhancer. The data suggest that estrogen regulates SK3 gene expression through interactions between ERalpha and Sp1. (C) 2003 Elsevier Science (USA). All rights reserved.