A Second Promoter for the Human KCl Cotransporter 1 (KCC1) Gene Drives Differential Expression of a Variant Isoform in Sickle Versus Normal Reticulocytes.
Authors: Anderson Kathleen , Crable Scott C. , Hammond Suzan M. , Joiner Clinton H. , Gallagher Patrick G. 
Published: October 2019

Abstract
The K+Cl- cotransporter plays a significant role in the maintenance of red cell volume. During cellular maturation, this cotransporter actively moves K+ and Cl- out of the cell. The accompanying movement of water results in dehydration and shrinking of the red cell. Because KCl cotransporter activity is higher in sickle compared to normal reticulocytes, it has been considered a potential modifier gene for sickle cell disease. We have evidence for expression of three KCC genes in human reticulocytes and have investigated the promoter for KCC1. While the expression of the principal KCC1 transcript did not differ in SS compared to normal reticulocytes, we now describe an alternative transcript of the KCC1 gene emanating from a second promoter and exhibiting a restricted tissue distribution. Investigation of the EST databases revealed spliced ESTs corresponding to the use of four distinct N-terminal exons in the KCC1 gene, each reported multiple times in the dataset. Primers were developed for these 5′ regions (exon1, 1a, 1b, and 1c) and used in an RT-PCR reaction with human reticulocyte RNA. The exon1 form and the exon1b variant were expressed. When the relative levels of these forms were compared, expression of the exon1 transcript was unchanged, while significantly higher levels of the exon1b variant was evident in the AA reticulocyte RNA compared to numerous SS samples. In an analysis of seven other human tissue samples, the exon1b isoform was highly expressed in kidney, lung, and heart, while the KCC1ex1 transcript was expressed at a constant level in all tissues. Although the transcript for this variant could arise from the KCC1 promoter we have previously characterized, the pattern of expression suggested control from a second promoter. A 915bp region corresponding to −787 to +128 was isolated and cloned into a reporter construct to test for promoter activity. This clone was compared with KCC1 promoter constructs in transient transfection assays. The exon1b construct not only exhibited promoter activity by directing high levels of luciferase expression in K562 cells, it also demonstrated tissue-specificity with a low level of activity in Jurkat cells. This recapitulates the endogenous levels detected by RT-PCR analysis of these cell lines. The −787/+128 exon1b construct is also 3-fold more active than the ubiquitously expressed exon1 promoter. To identify the control elements for this promoter, we produced a series of deletion constructs; the smallest construct contained 181bp. No reduction in reporter gene activity was evident, indicating the major regulatory elements lie very close to this promoter. Since exon1 encodes 39aa and exon1b only 7aa, the use of this smaller first exon effectively produces an N-terminal truncation in the protein. Studies with the mouse KCC1 cDNA have demonstrated that proteins produced by an N-terminal truncation are not only inactive for K+Cl- cotransport, but also function as dominant negative regulators of a full-length KCC1 protein. High level expression of this variant in AA cells compared to SS cells would therefore be consistent with the low reported activity in the AA reticulocytes. Induction or modulation of the expression of the KCC1ex1b variant may be an key factor in the control of red cell hydration.