CLONING AND CHARACTERIZATION OF THE PROMOTER FOR A POTASSIUM CHANNEL EXPRESSED IN HIGH-FREQUENCY FIRING NEURONS

The Kv3.1 potassium channel is expressed in neurons that generate trai ns of high frequency action potentials in response to synaptic inputs. To understand the mechanisms underlying the regulation and restricted expression pattern of the Kv3.1 gene, we have cloned and characterize d its promoter. We first isolated a 5.3-kilobase pair fragment of the Kv3.1 5'-flanking region. When linked to the chloramphenicol acetyltra nsferase reporter gene, this fragment was found to be active in the un differentiated PC12 cell line, a neuron-like cell line, but not in a f ibroblast cell line. By carrying out a series of deletion analyses in undifferentiated PC12 cells, we have localized the essential promoter region to a highly GC-rich region containing four Sp-1 binding sites. Similar deletion analysis in NIH3T3 cells suggests that multiple silen cing elements and enhancing element(s) are involved in the cell type-s pecific expression of this gene. Further regulatory elements, includin g one cyclic AMP/calcium response element (CRE) and one Ap-1 element w ere found in the upstream region of the promoter. Using a stable undif ferentiated PC12 cell line transfected with the Kv3.1 B'-flanking regi on, we determined that promoter activity is enhanced by a cAMP analog and a calcium ionophore. Deletion of the CRE-like element at position -252 eliminated the enhancement of promoter activity by cAMP, and mobi lity shift assays confirmed that the Kv3.1 CRE sequence binds both a n uclear factor in undifferentiated PC12 cells and recombinant CRE bindi ng protein. Our results suggest that the transcription of the Kv3.1 ch annel may be regulated by neurotransmitters that elevate cAMP levels i n neurons.