Electrophysiological characterization of contact sites in brain mitochondria.

From morphological and biochemical studies it has been recognized that the regions where the outer and inner membranes of mitochondria come in close contact (contact sites) can be the route mechanism through which mitochondria interact directly with the cytoplasm. We have studied these regions electrophysiologically with the patch clamp technique, with the aim of understanding if this direct interaction is mediated by high conductance ion channels similar to the channel already detected in the inner membrane of mitochondria (Sorgato M. C., Keller, B. U., and Stühmer, W. (1987) Nature 330, 498-500). Contact sites isolated from rat brain mitochondria were thus incorporated into liposomes subsequently enlarged sufficiently to be patch clamped. This study shows that these particular fractions contain ion channels with conductances ranging from approximately 5 picosiemens to 1 nanosiemens (in symmetrical 150 mM KCl). Most of these channels are not voltage-dependent and can be open at physiological potentials sustained by respiring mitochondria. The lack of voltage sensitivity seems not to be the outcome of methodological artifacts, as voltage-gated channels are detected in giant liposomes containing either the outer mitochondrial membrane or a partially purified fraction of the inner mitochondrial membrane. These data therefore indicate that channels present in mitochondrial contact sites have properties which render them amenable to perform several of the functions hypothesized for these regions, particularly that of translocating macromolecules from the cytoplasm to the matrix of mitochondria

Molecular basis of altered excitability in Shaker mutants of Drosophila melanogaster.

Mutations in the Shaker (Sh) locus of Drosophila melanogaster have differing effects on action potential duration and repolarization in neurons as well as on A-type K+ channels (I(A)) in muscle. The molecular basis of three exemplary Sh alleles (Sh(KS133), Sh(E62) and Sh5) has been identified. They are point mutation in the Sh transcription unit expressing aberrant voltage-gated A-type K+ channels. Replicas of each mutation have been introduced by in vitro mutagenesis into Sh cDNA. The expression of in vitro transcribed mutant Sh cRNA in Xenopus laevis oocytes reproduced the specific phenotypic traits of each Sh allele. The lack of I(A) in Sh(KS133) is due to a missense mutation within a sequence motif occurring in all hitherto characterized voltage-gated K+ channel forming proteins. The reduction of I(A) in Sh(E62) is due to a mutation in an AG acceptor site. The intervening sequence between exon 19 and 20 is not spliced in Sh(E62) RNA. As a consequence Sh(E62) flies do not contain the full complement of Sh K+ forming proteins. Finally, the Sh5 mutation leads to an altered voltage dependence of K+ channel activation and inactivation as well as to an accelerated rate of recovery from inactivation. This is due to a missense mutation altering the amino acid sequence of the proposed transmembrane segment S5 of the Sh K+ channels. Segment S5 is located adjacently to the presumed voltage sensor of voltage-gated ion channels. The results explain the altered properties of excitable cells in Sh mutants and provide a general model for the possible role of A-type K+ channels in modulation action potential profiles

Osteopenia Due to Enhanced Cathepsin K Release by BK Channel Ablation in Osteoclasts

BACKGROUND: The process of bone resorption by osteoclasts is regulated by Cathepsin K, the lysosomal collagenase responsible for the degradation of the organic bone matrix during bone remodeling. Recently, Cathepsin K was regarded as a potential target for therapeutic intervention of osteoporosis. However, mechanisms leading to osteopenia, which is much more common in young female population and often appears to be the clinical pre-stage of idiopathic osteoporosis, still remain to be elucidated, and molecular targets need to be identified. METHODOLOGY/PRINCIPAL FINDINGS: We found, that in juvenile bone the large conductance, voltage and Ca(2+)-activated (BK) K(+) channel, which links membrane depolarization and local increases in cytosolic calcium to hyperpolarizing K(+) outward currents, is exclusively expressed in osteoclasts. In juvenile BK-deficient (BK(-/-)) female mice, plasma Cathepsin K levels were elevated two-fold when compared to wild-type littermates. This increase was linked to an osteopenic phenotype with reduced bone mineral density in long bones and enhanced porosity of trabecular meshwork in BK(-/-) vertebrae as demonstrated by high-resolution flat-panel volume computed tomography and micro-CT. However, plasma levels of sRANKL, osteoprotegerin, estrogene, Ca(2+) and triiodthyronine as well as osteoclastogenesis were not altered in BK(-/-) females. CONCLUSION/SIGNIFICANCE: Our findings suggest that the BK channel controls resorptive osteoclast activity by regulating Cathepsin K release. Targeted deletion of BK channel in mice resulted in an osteoclast-autonomous osteopenia, becoming apparent in juvenile females. Thus, the BK(-/-) mouse-line represents a new model for juvenile osteopenia, and revealed the BK channel as putative new target for therapeutic controlling of osteoclast activity