The emerging picture of mitochondrial membrane channels.
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Intracellular Membranes↗
Biomedical subjects
H Tedeschi
Publications and source records attributed to H Tedeschi.
Voltage activation of heart inner mitochondrial membrane channels.
The patch clamp records obtained from mitoplast membranes prepared in the presence of a calcium chelator generally lack channel activity. However, multiconductance channel (MCC) activity can be induced by membrane potentials above +/- 60 mV [Kinnally et al., Biochem. Biophys. Res. Commun. 176, 1183-1188 (1991)]. Once activated, the MCC activity persists at all voltages. The present report characterizes the activation by voltage of multiconductance channels of rat heart inner mitochondrial membranes using patch-clamping. In some membrane patches, the size of single current transitions progressively increases with time upon application of voltage. The inhibitor cyclosporin has also been found to decrease channel conductance in steps. The results suggest that voltage-induced effects which are inhibited by cyclosporin A are likely to involve either an increase in effective pore diameter or the assembly of low-conductance units. In activated patches, we have found at high membrane potentials (e.g., 130 mV) changes in conductance as high as 5 nS occurring in large steps (up to 2.7 nS). These were generally preceded by a smaller transition. Similar results were obtained less frequently at lower voltages. These results can be explained on the assumption that once assembled the channels may act in unison.
Animals↗
Modified supraorbital craniotomy: technical note.
The authors present a surgical approach that incorporates the frontal sinus and extends a supraorbital craniotomy to include the lateral orbital rim and zygoma. The craniotomy provides wide exposure of the anterior fossa, orbit, ipsilateral middle fossa, and cavernous sinus. The procedure can be performed easily, and the bone flaps can be secured rapidly back into the anatomical position at the time of closure. This modified supraorbital craniotomy is ideal for large benign lesions originating along the sphenoid wing or orbit that expand into the anterior fossa.
Craniotomy↗
Microsurgical anatomy of acoustic neuroma.
Because acoustic neuromas most frequently arise in the posteriorly placed vestibular nerves, they usually displace the facial and cochlear nerves anteriorly (Figs. 11, 12, and 13). The facial nerve is stretched around the anterior half of the tumor capsule. Variability in the direction of growth of the tumor arising from the vestibular nerves may result in the facial nerve being displaced, not only directly anteriorly, but also anterior-superiorly or anterior-inferiorly. The nerve is infrequently found on the posterior surface of the tumor. Because the facial nerve always enters the facial canal at the anterior-superior quadrant of the lateral margin of the meatus, it is usually easiest to locate it here, rather than at a more medial location where the degree of displacement of the nerve is more variable. The cochlear nerve also lies anterior to the vestibular nerve and is most frequently stretched around the anterior half of the tumor. The strokes of the fine dissecting instruments used in removing the tumor should be directed along the vestibulocochlear nerve from medial to lateral rather than from lateral to medial because traction medially may tear the tiny filaments of the cochlear nerve at the site where these filaments penetrate the lateral end of the meatus to enter the cochlea. The landmarks that are helpful in identifying the facial and vestibulocochlear nerves at the brain stem on the medial side of the tumor have been reviewed. These nerves, although distorted by tumor, can usually be identified on the brain stem side of the tumor at the lateral end of the pontomedullary sulcus, just rostral to the glossopharyngeal nerve and just anterior-superior to the foramen of Luschka, flocculus, and choroid plexus protruding from the foramen of Luschka. After the facial and vestibulocochlear nerves are identified on the medial and lateral sides of the tumor, the final remnants of the tumor are separated from the intervening segment of the nerves. In the three approaches to the meatus and cerebellopontine angle--retrosigmoid, translabyrinthine, and middle fossa--a communication may be established between the subarachnoid space and the mastoid air cells that requires careful closure to prevent a cerebrospinal fluid leak.
Brain Stem↗
Selective effect of inhibitors on inner mitochondrial membrane channels.
The effect of amphiphilic cationic drugs on the channel activity of the mitochondrial inner membrane was examined with patch-clamp techniques. The therapeutic drugs amiodarone, propranolol and quinine reduced the probability of being open for the multiconductance channel (MCC) activity (levels from 30 pS to over 1 nS). While amiodarone decreased the probability of being open for the voltage dependent approximately 100 pS channel, it increased the conductance 42 +/- 20% (mean +/- SD, n = 6) with no significant change in mean open time. Similar results were obtained with propranolol. These data indicate that the approximately 100 pS channel is distinct from MCC activity.
Amiodarone↗
Identification of anion and cation pathways in the inner mitochondrial membrane by patch clamping of mouse liver mitoplasts.
Alkalinization of the matrix side of the mitochondrial inner membrane by pH shifts from 6.8 to 8.3 caused a reversible increase in current of 3.2 +/- 0.2 pA (mean +/- SE, n = 21) at +/- 40 mV measured using patch-clamp techniques. The current increase was reversed in a graded fashion by the addition of Mg2+ as well as a reduction in pH. Detection of single-channel events was done at 0.5, 1 and 2 M KCl. The single-channel amplitude in 0.15 M KCl corresponds to approximately 15 pS. Reversal potentials derived from whole patch currents indicated that the inner mitochondrial membrane was primarily cation selective at pH 6.8 with a PK/PCl = 32 (n = 6). Treatment with alkaline pH (8.3) increased the current and anion permeability (PK/PCl = 16, n = 6). The membrane becomes completely cation selective when low concentrations (12 microM) of the drug propranolol are added. The amphiphilic drugs amiodarone (4 microM), propranolol (70 microM) and quinine (0.6 mM) blocked almost all of the current. The pH-dependent current was also inhibited by tributyltin. These results are consistent with the presence of two pathways in the inner mitochondrial membrane. One is cation selective and generally open and the other is anion selective and induced by alkaline pH. The alkaline pH-activated channel likely corresponds to the inner membrane anion channel postulated by others from suspension studies.
Amiodarone↗
Anatomic examination of a case of open trigeminal nucleotomy (nucleus caudalis dorsal root entry zone lesions) for facial pain.
Nucleus caudalis dorsal root entry zone lesions (open trigeminal nucleotomy) are a surgical procedure which can achieve pain control without major complications in the difficult clinical setting of deafferentation-type facial pain. Two patients are reported, who had relief of pain, but also experienced neurological complications. One patient succumbed to pulmonary complications, which provided the opportunity for anatomic analysis of the lesioned area, which is discussed in detail. Potential modifications of the surgical technique are suggested.
Afferent Pathways↗
Properties of channels in the mitochondrial outer membrane.
Patch-clamping studies with native outer mitochondrial membranes show a complex behavior. In the range of potentials in which the polarity of the pipette is positive, the behavior resembles that of VDAC incorporated into bilayers. Accordingly, there is a decrease in conductance with voltage. An involvement of VDAC is also supported by responses of the patches to the presence of polyanion or treatment with succinic anhydride, both of which affect VDAC. In contrast, in the negative range of potential, the conductance of the patches generally increases with the magnitude of the voltage. The increase in conductance shows a biphasic time course which is consistent with a model in which channels are first activated (first phase) and then assembled into larger high-conductance channels (second phase). A variety of experiments support the notion that an assembly takes place. The time course of the conductance increase is consistent with formation of the second-phase channels from 6 +/- 1 subunits.
Animals↗
Mitochondrial channel activity studied by patch-clamping mitoplasts.
Patch-clamping mitoplasts, we have observed a complex pattern of conductance transitions. This report discusses primarily the 45, 120-150, 350, and 1,000 pS transitions.
Animals↗
Kinetics of voltage-induced conductance increases in the outer mitochondrial membrane.
The kinetics of the increase in conductance in the outer mitochondrial membrane induced by patch-clamping at various negative potentials (pipette inside negative) are reported. The changes are biphasic, a rapid increase is followed by a slowly developing larger change. The results can be predicted by a model in which an initial activation of channels is followed by their assembly into highly conducting channels. The model suggests that five to seven activated subunits form each high-conductance channel.
Animals↗
Evidence for a novel voltage-activated channel in the outer mitochondrial membrane.
Patch-clamp studies of the outer mitochondrial membrane indicate a voltage-dependent increase in conductance for potentials positive relative to the exterior of the mitochondrion. The time course of the conductance changes is consistent with an activation of channels. Voltage pulse experiments suggest that the activation phenomenon corresponds to assembly of the channels from subunits with disassembly occurring after recovery of the original conductance. Effects of temperature and concanavalin A on the voltage-induced conductances are also consistent with a channel assembly model.
Animals↗
Importance of the mitochondrial outer membrane channel as a model biological channel.
The channels of the mitochondrial outer membrane represent a useful model for studies into the mechanisms underlying phenomena of voltage-dependent gating and ion selectivity.
Intracellular Membranes↗
Channels in the mitochondrial outer membrane: evidence from patch clamp studies.
Patch clamp techniques were applied to outer mitochondrial membranes of giant mitochondria from mice kept on a cuprizone diet or to vesicles produced by fusing membranes derived from the outer membrane of Neurospora mitochondria. In the negative range of potentials the conductances decreased with increases in the magnitude of voltage, suggesting the closing of channels. Experiments in which mitochondria were treated with the polyanion polymethacrylate maleate styrene (1:2:3) or succinic anhydride suggest that the channels correspond to VDAC. Although sometimes conductance also decreased with increasing potential over a narrow range of positive potentials, more commonly the conductances increased. Although this phenomenon may represent a detachment of the patch, the changes in conductance are reversible, suggesting that they correspond to the formation or the opening of channels.
Electric Conductivity↗
Patch clamping the outer mitochondrial membrane.
Intact giant mitochondria isolated from the liver of mice fed a diet containing cuprizone were studied using patch microelectrodes. The current-voltage curves were nonlinear, suggesting the presence of voltage-sensitive channels. In the negative range of voltage, the channels appear to close with increasing magnitude of the voltage. The dependence of the conductance on voltage is similar to that of the outer membrane channels (VDAC) studied in planar bilayers. Occasionally, over a narrow range of positive potentials, the conductance also decreases as in the bilayer studies. However, more frequently the conductance increases sharply in a completely reversible manner at potentials greater than 10 to 20 mV. The increase in conductance with voltage may be interpreted as a major rearrangement of membrane components. Qualitatively comparable results were obtained using fused outer membranes isolated from Neurospora mitochondria. The behavior of VDAC is affected by treatment with succinic anhydride or the polyanion, polymethacrylate, maleate, styrene (1:2:3). We have found similar effects in the negative range of potentials in patches from giant mitochondria treated in the same fashion.
Animals↗
Protonmotive force and photophosphorylation in single swollen thylakoid vesicles.
Swollen vesicles generally 40 micron in diameter were prepared from spinach chloroplasts. These vesicles appear to originate from thylakoids. The present study reports results obtained with individual vesicles using micromanipulative procedures. The electric potential across the membrane was measured with microelectrodes and the pH of the internal space was calculated from the fluorescence of the pH indicator pyranine. The individual vesicles photophosphorylate as measured with luciferin-luciferase. Impalement with microelectrodes did not affect the ability of individual vesicles to photophosphorylate. However, there was no significant membrane potential either with continuous illumination or light flashes. In contrast, we found a delta pH of 3.7 under photophosphorylative conditions and the incubation with the appropriate buffers blocked photophosphorylation presumably by preventing formation of a pH gradient. We propose that, in these vesicles, the membrane potential plays no role in photophosphorylation, whereas a pH gradient is obligatory.
Adenosine Triphosphate↗
The internal concentration of K+ in isolated rat liver mitochondria.
A simple osmotic method has been developed to determine the internal K+ concentration of mitochondria by determining the concentration of external K+ at constant osmotic pressure at which metabolically inhibited mitochondria neither shrink nor swell. This concentration has been found to correspond to approx. 80-85 mM in freshly isolated mitochondria and considerably lower after additional centrifugation procedures. Since mitochondria are in osmotic equilibrium with the suspending medium (in this case, 0.32 osmolal), and K+ is the primary exchangeable internal ion, a significant proportion of the internal osmotic pressure must be exerted by the sucrose. Results for experiments determining internal K+ after centrifuging mitochondria at various G values confirm the reports of Sitaramam et al. (Sitaraman, V. and Sarma, M.K.J. (1981) Proc. Natl. Acad. Sci. USA 78, 3441-3445 and Sambasivarao, D. and Sitaramam, V. (1983) Biochim. Biophys. Acta 722, 256-270) that centrifugation induces the entry of sucrose in mitochondria isolated in a sucrose medium.
Animals↗
Assay of ATP synthesis using single giant mitochondria.
Two assays of the capacity of single giant mitochondria or mitoplasts to phosphorylate ATP from Pi and ADP have been developed. One depends on the placement of a single mitochondrion next to a glycerinated myofibril, by micromanipulation. With the appropriate controls, contraction of the myofibril serves as an indication of ATP synthesis. The other assay similarly requires the isolation of one single mitochondrion but the assay of ATP synthesized uses the luciferin-luciferase reaction with a conventional photometric system. With the latter, we found that either impalement with microelectrodes or electrophoretic microinjection of two dyes, Lucifer Yellow CH or pyranine , into the inner space have no effect on the phosphorylative capacity of mitochondria or mitoplasts. The electric potential across the mitochondrial membrane was monitored during the assay and found to be small and generally positive inside.
Adenosine Triphosphate↗
Protonmotive force in giant mitochondria.
The accompanying communication [Eur. J. Biochem. 141 (1984) 1-4] indicates that the microinjection of the pH fluorescent indicator pyranine (8-hydroxy-1,2,6- pyrenesulfonate ) into giant mitochondria or mitoplasts does not affect their ability to carry out oxidative phosphorylation. The dye can therefore be used as a quantitative indicator of internal mitochondrial pH. We found that activation of metabolism in rotenone-inhibited giant mitochondria by the addition of succinate produces an internal pH change corresponding to a pH shift of 0.3 to the alkaline range, approximately the same value found previously for conventional rat liver mitochondria.
Animals↗