Localization and trafficking of cardiac voltage-gated potassium channels.

The proper trafficking and localization of cardiac potassium channels is profoundly important to the regulation of the regionally distinct action potentials across the myocardium. These processes are only beginning to be unravelled and involve modulators of channel synthesis and assembly, post-translational processing, various molecular motors and an increasing number of modifying enzymes and molecular anchors. The roles of anchoring proteins, molecular motors and kinases are explored and recent findings on channel internalization and trafficking are presented.

Characterization of a surface modified dry powder inhalation carrier prepared by "particle smoothing".

Atomic force microscopy (AFM) was used to investigate drug-carrier interactions between beclometasone dipropionate (BDP) and a series of untreated and modified lactose surfaces. This quantitative information was correlated with bulk characterization methods and an in-vitro study. Modified lactose surfaces were prepared using a proprietary process referred to as "particle smoothing" to obtain smooth carrier surfaces with or without the presence of magnesium stearate. The engineering of lactose carrier surfaces using the particle smoothing process resulted in significant differences in surface morphology when compared with the "as supplied" starting material. The energy of separation, between BDP and lactose samples, determined by AFM suggested similar lognormal distributions with a rank decrease in median separation energy (e(0.5)) (26.7, 20.6 and 7.7 microJ for untreated, particle-smoothed and particle-smoothed with magnesium stearate, respectively). A series of in-vitro twin stage impinger studies showed good correlation with the AFM separation energy measurements. The mean fine particle dose increased for the two processed lactose samples, with a significant increase for the lactose processed with magnesium stearate, 102.0+/-16 microg compared with 24.2+/-10.7 microg for the untreated lactose. Thus, the AFM presents as a possible pre-formulation tool for rapid characterization of particle interactions.

Powder compaction properties of sodium starch glycolate disintegrants.

The compaction behavior of three "as supplied" commercially available grades of sodium starch glycolate (SSG), Explotab, Primojel, and Vivastar P, was investigated at compression speeds of 0.17 and 30 mm/sec. The results suggested that the three "as supplied" materials exhibit different compression and compaction behavior. Primojel and Explotab exhibited similar compactibility, whereas Vivastar P produced compacts of poor integrity. This behavior was not mirrored in the compressibility of the powders, where Vivastar P and Explotab exhibited similar performance. The materials were studied using x-ray diffraction, scanning electron microscopy, Carr's compressibility index, and swelling volume. In terms of material characteristics, all the products exhibited similar swelling in water. Primojel and Explotab retained most of the crystallographic order from the parent potato starch and exhibited comparable particle surface topographies. Vivastar P contained the lowest moisture level. However, it is not clear if the poor compactibility of Vivastar P is due to differences in moisture content, the reduced surface topography, or subtle differences in the SSG polymer structures (substitution, cross-linking, and crystallinity). Overall, even though the three commercial grades of sodium starch glycolate are successfully used as disintegrants, they do exhibit differences in their "as supplied" powder mechanical properties: Primojel and Explotab exhibit similar compactibility, whereas Vivastar P is poorly compactable but exhibits similar compressibility to Explotab. These observations may have implications when formulating poorly compactable or moisture-sensitive drugs.

Chemical characterisation of sodium starch glycolate particles.

The internal and surface chemical compositions of three sodium starch glycolate (SSG) products, Explotab, Primojel and Vivastar P were studied using scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), time of flight secondary ion mass spectrometry (TOF-SIMS) and 23Na nuclear magnetic resonance spectroscopy (NMR). The surfaces of Explotab and Primojel contained very distinct features containing Na and Cl, however, Primojel also contained features which contained Na which may reflect the presence of Na glycolate and/or Na citrates. Vivastar P contained relatively few surface Cl containing features. Analysis of cross-sections of the particles showed that Na appeared to be uniformly distributed throughout the particles of all the products. Additionally, there was a significant concentration of Cl in the periphery of Explotab and Primojel. In the case of Vivastar P, significant levels of Na and Cl were detected in the internal regions of the particles which, together with 23Na NMR, suggests that NaCl is uniformly distributed within Vivastar P. 23Na NMR also suggested that the ratio of organic Na to NaCl was considerably lower in Vivastar P than Primojel and Explotab. Overall, even though all these three products satisfy the pharmacopeial descriptions of SSG, these studies suggest that Primojel and Explotab exhibit different chemical compositions to Vivastar P. Since the three products studied are reported to be prepared from potato starch, the apparent differences in chemical composition probably reflect the different manufacturing processes used, however, batch to batch variations may account for some of the subtle differences.

Directional bonding in compacted microcrystalline cellulose.

The mechanical properties of compacts of microcrystalline cellulose (MCC) and silicified microcrystalline cellulose (SMCC) were evaluated by tensile testing, diametric compression testing, and compression testing. For tensile and compression testing, cubic specimens were carefully machined from MCC and SMCC compacts, and the tensile and compression strengths were evaluated both normal and parallel to the compaction direction. The cubic tensile strengths were compared to values obtained from the diametric compression test. The results obtained using the diametric compression test suggested compacts of SMCC exhibit greater strength than those of MCC. In addition, the cubes machined from compacts of MCC and SMCC exhibited directional strength; the direction normal to the compaction direction display ed the greater tensile strength, and the parallel direction had greater compression strength. The diametric compression test afforded strength values with reduced spread compared to the values collected from the cubic tensile test, suggesting that the errors involved in collecting diametric compression test data of compacts are less than those for the cubic tensile test. Analysis of the cubes using X-ray diffraction (XRD) suggested that they display directional structural anisotropy, with the direction normal to the compaction direction being more crystalline than the parallel direction. However, it is not clear whether the difference in the directional strength is solely a consequence of the increased crystallinity or a culmination of crystallographic and mechanical keying effects.

A discrete amino terminal domain of Kv1.5 and Kv1.4 potassium channels interacts with the spectrin repeats of alpha-actinin-2.

The interaction between the amino terminus of Kv1-type potassium channels and alpha-actinin-2 has been investigated. Using a combination of yeast two-hybrid analysis and in vitro binding assays, alpha-actinin-2 was found to bind to the N-termini of both Kv1.4 and Kv1.5 but not to the equivalent segments of Kv1.1, Kv1.2 or Kv1.3. Deletion analysis in the in vitro binding assays delineated the actinin binding region of Kv1.5 to between amino acids 73 and 148 of the channel. The Kv1.5 binding sites in alpha-actinin-2 were found to lie within actinin's internal spectrin repeats. Unlike the reported interaction between actinin and the NMDA receptor, calmodulin was found to have no effect on actinin binding to Kv1.5.

The mechanical properties of compacts of microcrystalline cellulose and silicified microcrystalline cellulose.

The mechanical properties of compacts of unlubricated microcrystalline cellulose and silicified microcrystalline cellulose were evaluated using the diametric tensile test. The results suggested that, under comparable testing conditions, compacts of silicified microcrystalline cellulose exhibited greater strength than those of microcrystalline cellulose. In addition to enhanced strength, silicified microcrystalline cellulose compacts exhibited greater stiffness and required considerably more energy for tensile failure to occur. Comparison of the data with that obtained for a dry blend of silicon dioxide/microcrystalline cellulose suggested that the functionality benefits of silicification were not due to a simple composite material model.

alpha-actinin-2 couples to cardiac Kv1.5 channels, regulating current density and channel localization in HEK cells.

Voltage-gated K(+) (Kv) channels are particularly important in the physiology of excitable cells in the heart and the brain. PSD-95 is known to cluster Shaker channels and NMDA receptors and the latter is known to couple through alpha-actinin-2 to the post-synaptic cytoskeleton [Wyszynski et al. (1997) Nature 385, 439-442], but the mechanisms by which Kv channels are linked to the actin cytoskeleton and clustered at specific sites in the heart are unknown. Here we provide evidence that Kv1.5 channels, widely expressed in the cardiovascular system, bind with alpha-actinin-2. Human Kv1.5 interacts via its N-terminus/core region and can be immunoprecipitated with alpha-actinin-2 both after in vitro translation and from HEK cells expressing both proteins. The ion channels and alpha-actinin-2 co-localize at the membrane in HEK cells, where disruption of the actin cytoskeleton and antisense constructs to alpha-actinin-2 modulate the ion and gating current density.

Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA-specific translational activation.

Genetic code differences prevent expression of nuclear genes within Saccharomyces cerevisiae mitochondria. To bridge this gap a synthetic gene, ARG8m, designed to specify an arginine biosynthetic enzyme when expressed inside mitochondria, has been inserted into yeast mtDNA in place of the COX3 structural gene. This mitochondrial cox3::ARG8m gene fully complements a nuclear arg8 deletion at the level of cell growth, and it is dependent for expression upon nuclear genes that encode subunits of the COX3 mRNA-specific translational activator. Thus, cox3::ARG8m serves as a mitochondrial reporter gene. Measurement of cox3::ARG8m expression at the levels of steady-state protein and enzymatic activity reveals that glucose repression operates within mitochondria. The levels of this reporter vary among strains whose nuclear genotypes lead to under- and overexpression of translational activator subunits, in particular Pet494p, indicating that mRNA-specific translational activation is a rate-limiting step in this organellar system. Whereas the steady-state level of cox3::ARG8m mRNA was also glucose repressed in an otherwise wild-type strain, absence of translational activation led to essentially repressed mRNA levels even under derepressing growth conditions. Thus, the mRNA is stabilized by translational activation, and variation in its level may be largely due to modulation of translation.

Time-dependent mitotic recombination in Saccharomyces cerevisiae.

The time-dependent appearance of prototrophic recombinants between heterologously located artificial repeats has been studied in Saccharomyces cerevisiae. While initial prototrophic colony numbers from independent cultures were highly variable, additional recombinants were found to arise daily at roughly constant rates irrespective of culture. These late-appearing recombinants could be accounted for neither by detectable growth on the selective media nor by delayed appearance of recombinants present at the time of selective plating. Significantly, at no time did the distributions of recombinants fully match those expected according to the Luria-Delbruck model and, in fact, after the first day, the distributions much more closely approximated a Poisson distribution. Prototrophic recombinants accumulated not only on the relevant selective medium, but also on media unrelated to the acquired prototrophy.

An examination of adaptive reversion in Saccharomyces cerevisiae.

Reversion to Lys+ prototrophy in a haploid yeast strain containing a defined lys2 frameshift mutation has been examined. When cells were plated on synthetic complete medium lacking only lysine, the numbers of Lys+ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. An examination of the distribution of the numbers of early appearing Lys+ colonies from independent cultures suggests that the mutations to prototrophy occurred randomly during nonselective growth. In contrast, an examination of the distribution of late appearing Lys+ colonies indicates that the underlying reversion events occurred after selective plating. No accumulation of Lys+ revertants occurred when cells were starved for tryptophan, leucine or both lysine and tryptophan prior to plating selectively for Lys+ revertants. These results indicate that mutations accumulate more frequently when they confer a selective advantage, and are thus consistent with the occurrence of adaptive mutations in yeast.

Allelic and ectopic interactions in recombination-defective yeast strains.

Ectopic recombination in the yeast Saccharomyces cerevisiae has been investigated by examining the effects of mutations known to alter allelic recombination frequencies. A haploid yeast strain disomic for chromosome III was constructed in which allelic recombination can be monitored using leu2 heteroalleles on chromosome III and ectopic recombination can be monitored using ura3 heteroalleles on chromosomes V and II. This strain contains the spo13-1 mutation which permits haploid strains to successfully complete meiosis and which rescues many recombination-defective mutants from the associated meiotic lethality. Mutations in the genes RAD50, SPO11 and HOP1 were introduced individually into this disomic strain using transformation procedures. Mitotic and meiotic comparisons of each mutant strain with the wild-type parental strain has shown that the mutation in question has comparable effects on ectopic and allelic recombination. Similar results have been obtained using diploid strains constructed by mating MATa and MAT alpha haploid derivatives of each of the disomic strains. These data demonstrate that ectopic and allelic recombination are affected by the same gene products and suggest that the two types of recombination are mechanistically similar. In addition, the comparison of disomic and diploid strains indicates that the presence of a chromosome pairing partner during meiosis does not affect the frequency of ectopic recombination events involving nonhomologous chromosomes.

Cytochrome P450 induction by phenobarbital (PB) is inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA): evidence that protein kinase C regulates induction.

The hepatic microsomal monooxygenase system was studied in hypophysectomized male rats exposed for 24 or 48 h to PB and/or TPA, an activator of kinase C. TPA attenuated basal and PB-induced levels of P450, aniline hydroxylase (ANH), ethylmorphine demethylase (EDM) and cytochrome c reductase. Hence, PB may effect induction via the inhibition of kinase C. Supporting this is spectral evidence that PB and TPA do not bind and the fact that TPA did not decrease P450 when co-incubated with O2 and NADPH. Hemin failed to increase P450 levels previously depressed by TPA indicating that TPA acts by lowering apocytochrome levels. This is consistent with its attenuation of PB-effected increases in hepatic RNA. TPA effects were associated with increased hepatic RNA and were blocked by puromycin.