Mobile phase flow-rate of 1. The PCR solution [1. The lanthanides, Th, U and Pu solution of appropriate concentrations were prepared after dilution with mobile phase.
Next Section Abstract The aim of this study was to determine whether functional heteromeric channels can be formed by co-assembly of rat SK3 rSK3 potassium channel subunits with either SK1 or SK2 subunits. These results suggest that hSK1 does not produce functional cell-surface assemblies with SK3.
Antibody-staining experiments suggested that hSK1 may reduce the number of functional SK3 subunits reaching the cell surface. Additional experiments showed that co-expression of the rat SK1 gene with SK3 also dramatically suppressed SK current. The pharmacology of the residual current was consistent with that of homomeric SK3 assemblies.
These results demonstrate interactions that cause changes in protein trafficking, cell surface expression, and channel pharmacology and strongly suggest heteromeric assembly of SK3 with the other SK channel subunits. In many neurons SK channels underlie some components of the post-spike after-hyperpolarization see e.
They also have important functions in non-neuronal tissues. Native SK channels have a characteristic pharmacology.
They can be blocked by the bee venom toxin apamin and several selective small molecule blockers that we have developed such as UCL that are active at nanomolar or subnanomolar concentrations 2 — 4. Molecular cloning studies have identified three closely related genes SK1, -2, and -3 which code for SK channel subunits in mammalian cells 5 — 7.
Further, both homomeric rSK2 and homomeric rSK3 channels can be blocked by either apamin or UCL at concentrations that are similar to those reported for native channels 8. The behavior of SK1 is different to that of other SK genes.
Initial expression studies of the human SK1 gene hSK1 showed that it can produce functional channels in the Xenopus oocyte expression system and these channels are insensitive to apamin at concentrations of up to nm 5. A few cells also produce an apamin-insensitive current component 9 More recent oocyte work suggests that, in fact, expression of the human SK1 gene also produces some apamin-sensitive channels in this system The reason for this behavior is unclear and it consequently remains unknown whether SK1 forms apamin-sensitive channels or apamin-insensitive channels, or both, in vivo.
Interestingly, the rat SK1 gene differs from its human counterpart because it does not produce functional channels when expressed alone in either oocytes or mammalian cell lines That co-assembly may occur is also suggested by the work of Ishii et al.
Following this observation we have recently shown that co-transfection of the rat SK1 and SK2 genes in HEK cells produces channels with a novel pharmacology, suggesting that subunits from these genes can also assemble to form functional heteromeric channels Interestingly, however, it has been reported that a fragment of SK3, when transfected into the Jurkat cell line, acts as a dominant negative, suppressing the endogenously expressed SK2 current This finding suggests that SK2 and SK3 may be able to form a heteromeric complex.
The principle aim of the present work was to examine, in more detail, the possibility that SK3 interacts with the other SK channel subunits; SK1 and SK2.
Len Kaczmarek and Dr. William Joiner Yale University. The human hSK1 clone accession number U was a generous gift from Prof. The protocol for transfection of HEK cells was varied slightly according to the precise aims of the experiment.
After incubation overnight the cells were re-plated into mm dishes and used within 2 days for recording. Cells used for immunohistochemistry were plated on mm square glass coverslips.PRODUCT.
1) Catfish Carl's Realistic Washes, proof Tattoo Wash, 2-oz.
and 4-oz. bottles, Hand-blended Lite Wash Pigment. The product is in plastic squeeze bottle with turn nozzle on top. 72 solution as a mobile phase, with 20 lL injection volume at a 73 ﬂow rate of mL min À1, and a detector set at nm wave- and the complete dissolution of free FA from the sam- ple (British Standards Institution, ), total FA, however, could be obtained by soaking the sample in a sulfuric acid Co 2+ 54 · 10 À3.
Recombinant mumps virus (rMuV) was derived from the Jeryl Lynn strain of MuV vaccine (Merck and Co., Inc., Westpoint, PA). Recombinant VSVN4CT1gag1 was derived from the Indiana serotype of rVSV. Construction and rescue of rMuVgag and rVSVN4CT1gag1 vectors. [1,2,3, C 4]Docosanoate was synthesized from stearyl bromide and ethyl-[U C 4]acetoacetate (Merck Sharp and Dohme).
ATP-citrate lyase was prepared from the livers of rats that had been starved for 2 days and then refed for 3 days with a high glucose diet. 3E Technology, Inc.
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