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SummaryWe report the characterization of rice OsHKT1 (Oryza sativa ssp. indica) homologous to the wheat K + / Na + -symporter HKT1. Expression of OsHKT1 in the yeast strain CY162 defective in K + -uptake restored growth at mM and mM concentrations of K + and mediated hypersensitivity to Na + . When expressed in Xenopus oocytes, rice OsHKT1 showed uptake characteristics of a Na + -transporter but mediated transport of other alkali cations as well. OsHKT1 expression was analysed in salt-tolerant rice Pokkali and salt-sensitive IR29 in response to external cation concentrations. OsHKT1 is expressed in roots and leaves. Exposure to Na + , Rb + , Li + , and Cs + reduced OsHKT1 transcript amounts in both varieties and, in some cases, incompletely spliced transcripts were observed. By in situ hybridizations the expression of OsHKT1 was localized to the root epidermis and the vascular tissue inside the endodermis. In leaves, OsHKT1 showed strongest signals in cells surrounding the vasculature. The repression of OsHKT1 in the two rice varieties during salt stress was different in various cell types with main differences in the root vascular tissue. The data suggest control over HKT expression as a factor that may distinguish salt stress-sensitive and stress-tolerant lines. Differences in transcript expression in space and time in different lines of the same species appear to be a component of ion homeostasis correlated with salt sensitivity and tolerance.
Since its discovery in a glioma cell line 15 years ago, mitochondrial BKCa channel (mitoBKCa) has been studied in brain cells and cardiomyocytes sharing general biophysical properties such as high K+ conductance (~300 pS), voltage-dependency and Ca2+-sensitivity. Main advances in deciphering the molecular composition of mitoBKCa have included establishing that it is encoded by the Kcnma1 gene, that a C-terminal splice insert confers mitoBKCa ability to be targeted to cardiac mitochondria, and evidence for its potential coassembly with β subunits. Notoriously, β1 subunit directly interacts with cytochrome c oxidase and mitoBKCa can be modulated by substrates of the respiratory chain. mitoBKCa channel has a central role in protecting the heart from ischemia, where pharmacological activation of the channel impacts the generation of reactive oxygen species and mitochondrial Ca2+ preventing cell death likely by impeding uncontrolled opening of the mitochondrial transition pore. Supporting this view, inhibition of mitoBKCa with Iberiotoxin, enhances cytochrome c release from glioma mitochondria. Many tantalizing questions remain open. Some of them are: how is mitoBKCa coupled to the respiratory chain? Does mitoBKCa play non-conduction roles in mitochondria physiology? Which are the functional partners of mitoBKCa? What are the roles of mitoBKCa in other cell types? Answers to these questions are essential to define the impact of mitoBKCa channel in mitochondria biology and disease.
Key pointsr Association of plasma membrane BK Ca channels with BK-β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BK Ca channel (mitoBK Ca ) by BK-β subunits is not established.r MitoBK Ca -α and the regulatory BK-β1 subunit associate in mouse cardiac mitochondria. r A large fraction of mitoBK Ca display properties similar to that of plasma membrane BK Ca when associated with BK-β1 (left-shifted voltage dependence of activation, V 1/2 = −55 mV, 12 µM matrix Ca 2+ ). r In BK-β1 knockout mice, cardiac mitoBK Ca displayed a low P o and a depolarized V 1/2 of activation (+47 mV at 12 µM matrix Ca 2+ ) r Co-expression of BK Ca with the BK-β1 subunit in HeLa cells doubled the density of BK Ca in mitochondria.r The present study supports the view that the cardiac mitoBK Ca channel is functionally modulated by the BK-β1 subunit; proper targeting and activation of mitoBK Ca shapes mitochondrial Ca 2+ handling.Abstract Association of the plasma membrane BK Ca channel with auxiliary BK-β1-4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBK Ca ) with regulatory subunits is unknown. We report that mitoBK Ca functionally associates with its regulatory subunit BK-β1 in adult rodent cardiomyocytes. Cardiac mitoBK Ca is a calcium-and voltage-activated channel that is sensitive to paxilline with a large conductance for K + of 300 pS. Additionally, mitoBK Ca displays a high open probability (P o ) and voltage half-activation (V 1/2 = −55 mV, n = 7) resembling that of plasma membrane BK Ca when associated with its regulatory BK-β1 Enrique Balderas is currently associated with the Nora Eccles Harrison Cardiovascular Research and Training Institute (CVRTI) at 3818 E. Balderas and others J Physiol 597.15subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BK Ca -α and its BK-β1 subunit. Mitochondria from the BK-β1 knockout (KO) mice showed sparse mitoBK Ca currents (five patches with mitoBK Ca activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V 1/2 of activation (+47 mV in 12 µM matrix Ca 2+ ). The reduced activity of mitoBK Ca was accompanied by a high expression of BK Ca transcript in the BK-β1 KO, suggesting a lower abundance of mitoBK Ca channels in this genotype. Accordingly, BK-β1subunit increased the localization of BKDEC (i.e. the splice variant of BK Ca that specifically targets mitochondria) into mitochondria by two-fold. Importantly, both paxilline-treated and BK-β1 KO mitochondria displayed a more rapid Ca 2+ overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBK Ca associates with its regulatory BK-β1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBK Ca channel that helps to maintain mitochondrial Ca 2+ homeostasis.
Calcium entering mitochondria potently stimulates ATP synthesis. Increases in calcium preserve energy synthesis in cardiomyopathies caused by mitochondrial dysfunction, and occur due to enhanced activity of the mitochondrial calcium uniporter channel. The signaling mechanism that mediates this compensatory increase remains unknown. Here, we find that increases in the uniporter are due to impairment in Complex I of the electron transport chain. In normal physiology, Complex I promotes uniporter degradation via an interaction with the uniporter pore-forming subunit, a process we term Complex I-induced protein turnover. When Complex I dysfunction ensues, contact with the uniporter is inhibited, preventing degradation, and leading to a build-up in functional channels. Preventing uniporter activity leads to early demise in Complex I-deficient animals. Conversely, enhancing uniporter stability rescues survival and function in Complex I deficiency. Taken together, our data identify a fundamental pathway producing compensatory increases in calcium influx during Complex I impairment.
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