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ATP binding cassette (ABC) transporters are a diverse superfamily of energy-dependent membrane translocases. Although responsible for the majority of transmembrane transport in bacteria, they are relatively uncommon in eukaryotic mitochondria. Organellar trafficking and import, in addition to quaternary structure assembly, of mitochondrial ABC transporters is poorly understood and may offer explanations for the paucity of their diversity. Here we examine these processes in ABCB10 (ABC-me), a mitochondrial inner membrane erythroid transporter involved in heme biosynthesis. We report that ABCB10 possesses an unusually long 105-amino acid mitochondrial targeting presequence (mTP). The central subdomain of the mTP (amino acids (aa) 36 -70) is sufficient for mitochondrial import of enhanced green fluorescent protein. The N-terminal subdomain (aa 1-35) of the mTP, although not necessary for the trafficking of ABCB10 to mitochondria, participates in the proper import of the molecule into the inner membrane. We performed a series of amino acid mutations aimed at changing specific properties of the mTP. The mTP requires neither arginine residues nor predictable ␣-helices for efficient mitochondrial targeting. Disruption of its hydrophobic character by the mutation L46Q/ I47Q, however, greatly diminishes its efficacy. This mutation can be rescued by cryptic downstream (aa 106 -715) mitochondrial targeting signals, highlighting the redundancy of this protein's targeting qualities. Mass spectrometry analysis of chemically cross-linked, immunoprecipitated ABCB10 indicates that ABCB10 embedded in the mitochondrial inner membrane homodimerizes and homo-oligomerizes. A deletion mutant of ABCB10 that lacks its mTP efficiently targets to the endoplasmic reticulum. Quaternary structure assembly of ABCB10 in the ER appears to be similar to that in the mitochondria.ABC 1 transporters comprise a large and diverse family of membrane translocases (1). Their function ranges from peptide transport to phospholipid flipping to anion channel formation. Members of the ABC transporter superfamily have been implicated in numerous human diseases (including cystic fibrosis (CFTR/ABCC7), adrenoleukodystrophy (ALDP/ABCD1), Zellweger's syndrome (PMP70/ABCD3), progressive familial intrahepatic cholestasis (SPGP/ABCB11), and Stargardt macular dystrophy (ABCR/ABCA4)) (2). The basic structure of ABC transporters is relatively well conserved and includes a hydrophilic ATP binding cassette and a hydrophobic membranespanning domain (3). Whereas the large members of the family contain two of each domain, the "half-transporters" contain one of each and are predicted to dimerize (3-6). Mammalian ABC transporters are found predominantly in the plasma membrane but are also known to play essential roles in a number of organelles, including the endoplasmic reticulum, peroxisome, and the mitochondrion (2). Mammalian mitochondrial ABC transporters have recently gained attention for their role in heme biosynthesis and iron sulfur cluster synthesis (7-12). According...

Under many different conditions activated plasma membrane ion channels give rise to changes in the extracellular concentration of the permeant ion(s). The magnitude and duration of these changes are dependent on the electrochemical driving force(s) on the permeant ion(s) as well as conductance, open time, and channel density. We have modeled the change in the extracellular [K+] due to efflux through Ca2+-activated K+ channels, mSlo, to determine the range of parameters that would give rise to measurable signals in the surrounding media. Subsequently we have used extracellular, K+-selective microelectrodes to monitor localized changes in [K+]ext due to efflux through mSlo channels expressed in Xenopus oocytes. The rapid changes in [K+] show a close fit with the predicted model when the time response of the ion-selective microelectrode is taken into account, providing proof of the concept. Measurement of the change in extracellular ion concentration with ion-selective microelectrodes provides a noninvasive means for functional mapping of channel location and density, as well as characterizing the properties of ion channels in the plasma membrane.

Amoeba proteus may drive a current through themselves, and this current may determine the direction of cytoplasmic streaming. Cytoplasm streams from the more contracted tail or uroid region to the extending pseudopods. The positions of the uroid and pseudopods change as the amoeba changes direction. The membrane potential of the amoeba may change with location in the cell. This voltage gradient may regulate the magnitude and direction of cytoplasmic streaming. Bingley and Thompson (1962), recorded from amoebae using intracellular microelectrodes and reported that the membrane potential of the uroid region was more negative than that of the pseudopods. In contrast, Nuccitelli et al. (1977), using a vibrating probe, observed that current entered the uroid region and exited through the pseudopods. They concluded that the uroid region was more depolarized than the pseudopods. We revisited this issue by recording membrane potential simultaneously from two regions of the amoeba and by recording ionic currents using the two‐electrode voltage clamp. Our data support the findings of Nuccitelli et al. Cytoplasm may stream from more depolarized to more hyperpolarized regions. Moreover, overshooting depolarizations of the cell may produce global cytoplasmic contractions and rounding of the amoeba.