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Is Kir6.1 a subunit of mitoK(ATP)?

ABSTRACT: The subunit composition of the mitochondrial ATP-sensitive K(+)-channel (mitoK(ATP)) is unknown, though some suspect a role for the inward rectifier, Kir6.1, based largely on antibody studies of heart mitochondria. To ascertain the molecular identity of mitoK(ATP) we therefore sought to purify this putative mitochondrial Kir6.1, and conclusively identify the subunits by mass spectrometry. Immunoblots, conducted with two commercially available antibodies, revealed two distinct signals in isolated heart mitochondria, of 51 and 48kDa, respectively. Localization was confirmed by either immuno-gold electron microscopy or by immunofluorescence. Each putative Kir6.1 species was extracted, purified, and identified by LC-MS/MS. The 51kDa band was identified as NADH-dehydrogenase flavoprotein 1, while the preponderant protein in the 48-kDa band was mitochondrial isocitrate dehydrogenase (NADP form). 1D-, 2D-, and native gel analyses were consistent with these assignments. The data suggest it is premature to assign Kir6.1 a role in mitoK(ATP) on the basis of immunoreactivity alone.

SUBMITTER: Foster DB

PROVIDER: S-EPMC2276631 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Is Kir6.1 a subunit of mitoK(ATP)?

Foster D Brian DB Rucker Jasma J JJ Marbán Eduardo E

Biochemical and biophysical research communications 20071207 3

The subunit composition of the mitochondrial ATP-sensitive K(+)-channel (mitoK(ATP)) is unknown, though some suspect a role for the inward rectifier, Kir6.1, based largely on antibody studies of heart mitochondria. To ascertain the molecular identity of mitoK(ATP) we therefore sought to purify this putative mitochondrial Kir6.1, and conclusively identify the subunits by mass spectrometry. Immunoblots, conducted with two commercially available antibodies, revealed two distinct signals in isolated ...[more]

PMID: 18068667

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Project description:Key pointsSpontaneous contractions are essential for normal lymph transport and these contractions are exquisitely sensitive to the KATP channel activator pinacidil. KATP channel Kir6.1 and SUR2B subunits are expressed in mouse lymphatic smooth muscle (LSM) and form functional KATP channels as verified by electrophysiological techniques. Global deletion of Kir6.1 or SUR2 subunits results in severely impaired lymphatic contractile responses to pinacidil. Smooth muscle-specific expression of Kir6.1 gain-of-function mutant (GoF) subunits results in profound lymphatic contractile dysfunction and LSM hyperpolarization that is partially rescued by the KATP inhibitor glibenclamide. In contrast, lymphatic endothelial-specific expression of Kir6.1 GoF has essentially no effect on lymphatic contractile function. The high sensitivity of LSM to KATP channel GoF offers an explanation for the lymphoedema observed in patients with Cantú syndrome, a disorder caused by gain-of-function mutations in genes encoding Kir6.1 or SUR2, and suggests that glibenclamide may be an appropriate therapeutic agent.AbstractThis study aimed to understand the functional expression of KATP channel subunits in distinct lymphatic cell types, and assess the consequences of altered KATP channel activity on lymphatic pump function. KATP channel subunits Kir6.1 and SUR2B were expressed in mouse lymphatic muscle by PCR, but only Kir6.1 was expressed in lymphatic endothelium. Spontaneous contractions of popliteal lymphatics from wild-type (WT) (C57BL/6J) mice, assessed by pressure myography, were very sensitive to inhibition by the SUR2-specific KATP channel activator pinacidil, which hyperpolarized both mouse and human lymphatic smooth muscle (LSM). In vessels from mice with deletion of Kir6.1 (Kir6.1-/- ) or SUR2 (SUR2[STOP]) subunits, contractile parameters were not significantly different from those of WT vessels, suggesting that basal KATP channel activity in LSM is not an essential component of the lymphatic pacemaker, and does not exert a strong influence over contractile strength. However, these vessels were >100-fold less sensitive than WT vessels to pinacidil. Smooth muscle-specific expression of a Kir6.1 gain-of-function (GoF) subunit resulted in severely impaired lymphatic contractions and hyperpolarized LSM. Membrane potential and contractile activity was partially restored by the KATP channel inhibitor glibenclamide. In contrast, lymphatic endothelium-specific expression of Kir6.1 GoF subunits had negligible effects on lymphatic contraction frequency or amplitude. Our results demonstrate a high sensitivity of lymphatic contractility to KATP channel activators through activation of Kir6.1/SUR2-dependent channels in LSM. In addition, they offer an explanation for the lymphoedema observed in patients with Cantú syndrome, a disorder caused by gain-of-function mutations in genes encoding Kir6.1/SUR2.

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Is Kir6.1 a subunit of mitoK(ATP)?

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Is Kir6.1 a subunit of mitoK(ATP)?

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