A mitochondrial megachannel resides in monomeric F1FO ATP synthase

Purified mitochondrial ATP synthase has been shown to form Ca2+-activated, large conductance channel activity similar to that of mitochondrial megachannel (MMC) or mitochondrial permeability transition pore (mPTP) but the oligomeric state required for channel formation is being debated. We reconstitute purified monomeric ATP synthase from porcine heart mitochondria into small unilamellar vesicles (SUVs) with the lipid composition of mitochondrial inner membrane and analyze its oligomeric state by electron cryomicroscopy. The cryo-EM density map reveals the presence of a single ATP synthase monomer with no density seen for a second molecule tilted at an 86o angle relative to the first. We show that this preparation of SUV-reconstituted ATP synthase monomers, when fused into giant unilamellar vesicles (GUVs), forms voltage-gated and Ca2+-activated channels with the key features of mPTP. Based on our findings we conclude that the ATP synthase monomer is sufficient, and dimer formation is not required, for mPTP activity.

Ion channels ofFasciola hepaticaincorporated into planar lipid bilayers

Ion channels are important target sites of anthelmintics, but little is known about those inFasciola hepatica. In this work, we applied a planar lipid bilayer technique to characterize the properties of single ion channels inF. hepatica. Under a 200/40 mMKCl gradient, a large conductance channel of 251 pS was observed in 18% of the membranes studied. The channel was selective to K+over Cl−with a permeability ratio of K+to Cl−(PK/PCl) of 4·9. Open state probability (Po) of the channel was less than 0·5 and dependent on voltage (−60~+40 mV) and Ca2+(~100 μM). The other two types of single channels observed in 11 and 5% of membranes, respectively, were a K+-permeable channel of 80 pS (PK/PCl=4·6) and a Cl−-permeable channel of 64 pS (PK/PCl=0·058). Open state probability of both channels showed little voltage dependence. The results indicate that distinct single channels of 60~251 pS are present in relative abundance and, in addition, that the planar lipid bilayer technique can be a useful tool for the study of single ion channels inF. hepatica.

Molecular identification of Ca2+-activated K+ channels in parotid acinar cells

We used molecular biological and patch-clamp techniques to identify the Ca2+-activated K+ channel genes in mouse parotid acinar cells. Two types of K+ channels were activated by intracellular Ca2+ with single-channel conductance values of 22 and 140 pS (in 135 mM external K+), consistent with the intermediate and maxi-K classes of Ca2+-activated K+ channels, typified by the mIK1 ( Kcnn4) and mSlo ( Kcnma1) genes, respectively. The presence of mIK1 mRNA was established in acinar cells by in situ hybridization. The electrophysiological and pharmacological properties of heterologously expressed mIK1 channels matched those of the native current; thus the native, smaller conductance channel is likely derived from the mIK1 gene. We found that parotid acinar cells express a single, uncommon splice variant of the mSlo gene and that heterologously expressed channels of this Slo variant had a single-channel conductance indistinguishable from that of the native, large-conductance channel. However, the sensitivity of this expressed Slo variant to the scorpion toxin iberiotoxin was considerably different from that of the native current. RT-PCR analysis revealed the presence of two mSlo β-subunits ( Kcnmb1 and Kcnmb4) in parotid tissue. Comparison of the iberiotoxin sensitivity of the native current with that of parotid mSlo expressed with each β-subunit in isolation and measurements of the iberiotoxin sensitivity of currents in cells from β1 knockout mice suggest that parotid acinar cells contain approximately equal numbers of homotetrameric channel proteins from the parotid variant of the Slo gene and heteromeric proteins composed of the parotid Slo variant in combination with the β4-subunit.

Activation currents, sperm entry and surface contractions in ascidian eggs

SummarySpermatozoa from the mollusc Ostrea edulis are capable of fusing to and entering de-chorionated ascidian eggs. During interaction they generate activation currents, comparable to the fertilisation currents induced by homologous spermatozoa. Activation currents are inward at − 80 mV, with a mean initial slope of 111 ± 124 pA/s for Ciona intestinalis eggs and 47 ± 25 pA/s for Phallusia mammillata eggs, while the mean peak currents are 2782 ± 1132 pA and 1523 ± 1668 pA, respectively. The fertilisation and activation currents reverse at a holding potential of 0 mV to + 20 mV, suggesting that oyster sperm and ascidian sperm gate the same channel precursor, a non-specific, large conductance channel described previously (Dale & DeFelice, 1984). In contrast to homologous fertilisation, the activation current is not followed by a polarised contraction of the egg surface, nor other signs of egg activation. Staining eggs with Hoechst 33342 after insemination shows the female nucleus and a single oyster sperm nucleus at the antipode. This suggests a specialised predetermined site at the vegetal pole for sperm entry. Homologous and heterologous spermatozoa delivered, in a large pipette, to localised areas of the egg surface generate fast inward currents of 200–2000 pA, but do not induce contraction of the egg surface. This shows that although channel precursors are located globally over the egg surface, channel activation does not necessarily trigger the contraction wave. Subsequent induction of both a fertilisation current and a contraction by homologous sperm added to the bath, implies a regionalised activation site with an accumulation of channel precursors and a ‘pacemaker’ for the initiation of the contraction wave.