Kcnma1 alternative splicing in mouse kidney: regulation during development and by dietary K+ intake

Abstract

The pore-forming α-subunit of the large-conductance K⁺ (BK) channel is encoded by a single gene, KCNMA1. BK channel-mediated K⁺ secretion in the kidney is crucial for overall renal K⁺ homeostasis in both physiological and pathological conditions. BK channels achieve phenotypic diversity by various mechanisms, including substantial exon rearrangements at seven major alternative splicing sites. However, KCNMA1 alternative splicing in the kidney has not been characterized. The present study aims to identify the major splice variants of mouse Kcnma1 in whole kidney and distal nephron segments. We designed primers that specifically cross exons within each alternative splice site of mouse Kcnma1 and performed real-time quantitative RT-PCR (RT-qPCR) to quantify relative abundance of each splice variant. Our data suggest that Kcnma1 splice variants within mouse kidney are less diverse than in the brain. During postnatal kidney development, most Kcnma1 splice variants at site 5 and the COOH terminus increase in abundance over time. Within the kidney, the regulation of Kcnma1 alternative exon splicing within these two sites by dietary K⁺ loading is both site and sex specific. In microdissected distal tubules, the Kcnma1 alternative splicing profile, as well as its regulation by dietary K⁺, are distinctly different than in the whole kidney, suggesting segment and/or cell type specificity in Kcnma1 splicing events. Overall, our data provide evidence that Kcnma1 alternative splicing is regulated during postnatal development and may serve as an important adaptive mechanism to dietary K⁺ loading in mouse kidney.NEW & NOTEWORTHY We identified the major Kcnma1 splice variants that are specifically expressed in the whole mouse kidney or aldosterone-sensitive distal nephron segments. Our data suggest that Kcnma1 alternative splicing is developmentally regulated and subject to changes in dietary K⁺.

Keywords: BK channel; Kcnma1; kidney; potassium; splice variants.

Graphical abstract

Figure 1.

Alternative splicing variants of mouse Kcnma1. A: a scheme showing Kcnma1 exon arrangements at splice sites 1–7. B: predicted amino acid sequences of Kcnma1 variants at splicing sites 1–7. Variants at site 7 were named by their last 3 amino acid sequences (underlined), and asterisks indicate the stop codon.

Figure 2.

Detection of specific Kcnma1 variants at 7 splice sites in the mouse brain and kidney. Total RNA was isolated from the adult male C57BL/6 brain and kidney. A: RT-PCR was performed by using exon-crossing primer pairs to capture splicing events at each specific site in mouse brain and whole kidney; 5 µL of PCR products were examined in 2% agarose gels to confirm amplicon sizes and primer specificity. B: real-time quantitative RT-PCR (RT-qPCR) was performed to estimate the relative abundances of splice variants at each site in 4 brains and 5–7 kidneys. Data are presented in pie charts as percentages of the total transcripts. The distribution profile of Kcnma1 transcripts at each site was compared between the brain and kidney by chi-square test, and the exact P values are shown.

Figure 3.

Expression of Kcnma1 splice variants in developing mouse kidneys. Real-time quantitative RT-PCR (RT-qPCR) was performed with total RNA of mouse kidneys harvested at postnatal days 0, 7, 14, and 28. A and B: the abundance of each Kcnma1 splice variant at site 5 (A) and site 7 (B) is shown as a percentage of actin. Data are presented as means ± SD of 3–5 mice for each data point. Two-way ANOVA was performed, and the exact P values for age and sex are shown in each panel.

Figure 4.

Dietary K⁺ intake differentially regulates the expression of Kcnma1 splice variants. Total RNA was isolated from kidneys of adult C57BL/6 mice kept on a control diet [Ctrl (○), 8 males (blue bars) and 7 females (salmon bars)] or a high-K⁺ (10% KCl) diet [HK (●), 7 males (blue bars) and 6 females (salmon bars)] for 9 days. A and B: the abundance of each splice variant at site 5 (A) or site 7 (B) of Kcnma1 was normalized to actin and shown as fold change ($2^{\Delta \Delta {\mathrm{C}}_{\mathrm{t}}}$, where C_t is threshold cycle) over the Ctrl mice of the same sex. Data are presented in scatter-dot plots with bars showing means ± SD. Statistical comparisons were analyzed by two-way ANOVA followed by an uncorrected Fisher’s least significant difference (LSD) test, with exact P values shown for each comparison.

Figure 5.

Kcnma1 splice variants in the distal tubule (DT). Adult male C57BL/6 mice were kept on a control (Ctrl, n = 4) or high-K⁺ (HK) diet (10% KCl, n = 3) for 9–10 days. DTs were manually dissected based on the unique bifurcation morphology. Total RNA was isolated for quantitative RT-PCR (RT-qPCR) analysis. A: AQP2 staining (red) of the connecting tubule (CNT)/cortical collecting duct (CCD) in an isolated DT. The nucleus was labeled with TO-PRO-3 (in blue). B: the relative abundance of Aqp2 (AQP2, a marker of the CNT and CCD) vs. Slc12a1 [NKCC2, a marker for the thick ascending limb (TAL)] was assessed to confirm DT enrichment. DTs were dissected from 4 Ctrl mice (◊) and 3 HK mice (♦). Whole kidney total RNA of adult male mice fed with the control diet (○) was included as a control. C: the relative abundance of splice variants at site 5 or 7 within Ctrl DTs is shown in pie charts and compared to that of the Ctrl whole kidney (shown in Fig. 2B) by chi-square test, and the P values are shown. D: the abundance of each splice variant at splicing site 5 or site 7 in DTs of HK mice was normalized to actin and shown as fold changes ($2^{\Delta \Delta {\mathrm{C}}_{\mathrm{t}}}$, where C_t is threshold cycle) over Ctrl mouse DTs. Data are presented in scatter-dot plots with bars showing means ± SD. Statistical comparisons were analyzed with two-way ANOVA followed by an uncorrected Fisher’s least significant difference (LSD) test, with exact P values shown in each comparison.