Regulation of Mouse Slo Gene Expression

Abstract

The large conductance, voltage-and Ca 2؉-activated K ؉ channel plays key roles in diverse body functions influenced by estrogen, including smooth muscle and neural activities. In mouse (m), estrogen up-regulates the transcript levels of its pore-forming ␣-subunit (Slo, KCNMA1), yet the underlying genomic mechanism(s) is(are) unknown. We first mapped the promoters and regulatory motifs within the mSlo 5-flanking sequence to subsequently identify genomic regions and mechanisms required for estrogen regulation. mSlo gene has at least two TATA-less promoters with distinct potencies that may direct mSlo transcription from multiple transcription start sites. These qualities mark mSlo as a prototype gene with promoter plasticity capable of generating multiple mRNAs and the potential to adapt to organismal needs. mSlo promoters contain multiple estrogen-responsive sequences, e.g. two quasiperfect estrogen-responsive elements, ERE1 and ERE2, and Sp1 sites. Accordingly, mSlo promoter activity was highly enhanced by estrogen and blocked by estrogen antagonist ICI 182,780. When promoters are embedded in a 4.91-kb backbone, estrogen responsiveness involves a classical genomic mechanism, via ERE1 and ERE2, that may be complemented by Sp factors, particularly Sp1. Simultaneous but not individual ERE1 and ERE2 mutations caused significant loss of estrogen action. ERE2, which is closer to the proximal promoter, up-regulates this promoter via a classical genomic mechanism. ERE2 strategic position together with ERE1 and ERE2 independence and Sp contribution should ensure mSlo estrogen responsiveness. Thus, the mSlo gene seems to have uniquely evolved to warrant estrogen regulation. Estrogen-mediated mSlo genomic regulation has important implications on long term estrogenic effects affecting smooth muscle and neural functions. . As a consequence, MaxiK channels are key regulators of vital body functions such as, blood flow, neural function, and uresis (1-3). MaxiK channels are formed by four pore-forming ␣-subunits (Slo) that can associate with regulatory ␤-subunits (4). The ␣-subunit is expressed from a single copy gene in all studied species known as KCNMA1 or Slo. In mouse, it is present in chromosome 14 and has 27 constitutive exons, which are evolutionarily conserved.Knock-out mouse models have highlighted the importance of Slo expression in blood pressure regulation (5), hearing (6), urinary bladder contraction (7), erectile function (8), and neurological disorders (9). Furthermore, a mutation in the human gene has been linked to coexistent generalized epilepsy and paroxysmal dyskinesia (10).Defining the mechanisms that rule Slo gene expression is key to understanding the basis of Slo channel function. Several publications in recent years show that Slo transcript levels can be under the control of sex hormones and pregnancy. For example, mSlo transcript levels were up-regulated with the advancement of pregnancy in mouse myometrium (11, 12) likely due to the change in sex hormone levels. Consistent with this idea, mSlo transcr...

Distinct Transcriptional Regulation of Human Large Conductance Voltage- and Calcium-activated K+ Channel Gene (hSlo1) by Activated Estrogen Receptor α and c-Src Tyrosine Kinase

“…of the fit). These values are significantly lower than those observed for mSlo1 (EC 50 of 0.4 nM for a 2.9 kb and 0.34 for a 4.9-kb construct) (21). Confirming the need for activated ER␣, the anti-estrogen ICI 182,780 successfully antagonized estrogenmediated up-regulation of hSlo1 transcription in a dose-dependent manner (Fig.…”

“…hSlo1 versus mSlo1 Regulation by Estrogen and Underlying Mechanisms-Although both hSlo1 (this work) and mSlo1 (21) genes are up-regulated by estrogen via activation of ER␣ (as assessed by lack of estrogenic effect in the absence of receptor and by the antagonistic action of the anti-estrogen ICI 182,780; Fig. 2), several important differences were made evident in the present studies: (i) estrogen regulation of hSlo1 is much more potent (ϳ6-fold) than mSlo1; (ii) opposite to mSlo1, the hSlo1 5-kb promoter lacks quasi-perfect EREs; (iii) the DNA-binding domain mutant of hER␣ (DBDM-hER␣) or knocking down Sp1 did not decrease estrogen-action on hSlo1 as it does in mSlo1, ruling out direct or tethered (via Sp1) genomic mechanisms of action on hSlo1 by activated-hER␣; and (iv) activated ER␣ utilizes a nongenomic/signaling mechanism to upregulate hSlo1 as opposed to the genomic mechanism underlying mSlo1 upregulation (21).…”

“…Surprisingly, when DBDM-hER␣ was expressed, the stimulatory effect of 1 nM estrogen on hSlo1 promoter activities was not decreased (as observed for mSlo1, (21)) but on the contrary, estrogen was able to stimulate transcription even further than that achieved by wild type hER␣. This was true for both, the 2.5-and 5-kb hSlo1 promoters.…”

“…hER␣ Activation Leads to Potent Transcription of hSlo1-We have previously shown that activation of ER␣ promotes transcription of mSlo1 through a genomic mechanism by directly binding to the DNA (21). To determine whether hER␣ employs the same mechanism to promote transcription of hSlo1, we studied two 5Ј constructs containing 2520 nt (2.5 kb) and 5040 nt (5 kb) upstream of the third possible ATG translation initiation codon (encoding Met-3) of hSlo1 (23).…”

“…Also, monomeric G-protein Rho pathways are involved in ER␣-mediated transcriptional activity * This work was supported, in whole or in part, by National Institutes of Health (20). In the case of mSlo1, a genomic mechanism defines the up-regulation by estrogen-activated ER␣, where ER␣ binds to quasi-perfect EREs in the mSlo1 promoter, a mechanism that may be complemented by Sp1 transcription factor (21).…”