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- W2000383450 abstract "We reported that in the mouse, cardiac mitoBKCa is likely encoded by Kcnma1 gene, and that a splice insert DEC could facilitate BKCa targeting to mitochondria in CHO cells. Here, we examined whether cardiac mitochondrial membranes contain BKCa protein by mass spectrometry, and whether BKCa-containing DEC can be observed in cardiac mitochondria of adult cardiomyocytes.We first isolated mitochondria from rat cardiomyocytes and corroborated by immunoblot the presence of a ∼125 kDa protein corresponding to Kcnma1 (n=3). To enrich for inner mitochondrial membranes, cardiac BKCa was immunoprecipitated from mitoplasts. Six unique BKCa peptides spanning the whole C-terminus were found. The most upstream peptide, YGGSYSAVSGR, is near S6 transmembrane region while the extreme C-terminal peptide, GGYSTPQTLANR, is located after the Ca2+ bowl. Corroborating the presence of BKCa in mitoplasts, patch clamp recordings showed a large-conductance 226 pS K+ channel. Next, weanalyzed DEC relative abundance by quantitative RT-PCR using RNA from isolated mouse cardiomyocytes and found that DEC transcript levels are similar to that of total BKCa (n=3). Transcripts of other splice inserts, STREX and SV27, were less abundant (∼75% and 0.1% of total BKCa, respectively) suggesting a predominant expression of BKCa-DEC variant in cardiomyocytes. Finally, rat adult cardiomyocytes were transduced using N-terminal cMyc-tagged constructs of BKCa-DEC, and of insertless-BKCa and BKCa-SV27 (controls). Insertless-BKCa and BKCa-SV27 were labeled at the plasma membrane in non-permeabilized conditions. After permeabilization, intracellular signals did not colocalize with mitotracker (n=3). In contrast, BKCa-DEC signals were undetectable in non-permeabilized conditions but readily detected after permeabilization when they can be found colocalized with mitotracker to a similar extent as native BKCa (n=3). We conclude that BKCa exists in cardiac mitochondria and that mitoBKCa predominantly consists of a DEC-containing isoform which facilitates its targeting to cardiac mitochondria. Supported by NIH and AHA." @default.
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- W2000383450 date "2012-01-01" @default.
- W2000383450 modified "2023-10-17" @default.
- W2000383450 title "Targeting BKCa Channel to Mitochondria of Adult Cardiomyocytes" @default.
- W2000383450 doi "https://doi.org/10.1016/j.bpj.2011.11.3749" @default.
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