The rat SK1 gene (rSK1) does not form functional Ca2+‐activated potassium channels when expressed alone in mammalian cell lines. Using a selective antibody to the rSK1 subunit and a yellow fluorescent protein (YFP) tag we have discovered that rSK1 expression produces protein that remains largely at intracellular locations. We tested the idea that rSK1 may need an expression partner, rSK2, in order to form functional channels. When rSK1 was co‐expressed with rSK2 in HEK 293 cells it increased the current magnitude by 77 ± 34 % (as compared with cells expressing rSK2 alone). Co‐expression of rSK1 with rSK2 also changed the channel pharmacology. The sensitivity of SK current to block by apamin was reduced ~16‐fold from an IC50 of 94 pm (for SK2 alone) to 1.4 nm (for SK2 and SK1 together). The sensitivity to block by UCL 1848 (a potent small molecule blocker of SK channels) was similarly reduced, ~26‐fold, from an IC50 of 110 pm to 2.9 nm. These data clearly demonstrate that rSK1 and rSK2 subunits interact. The most likely explanation for this is that the subunits are able to form heteromeric assemblies.

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. First, to determine whether rSK3 could co-assemble with rSK2 we created rSK3VK (an SK3 mutant insensitive to block by UCL 1848). When rSK3VK was co-expressed with rSK2 the resulting currents had an intermediate sensitivity to UCL 1848 (IC 50 of ϳ5 nM compared with 120 pM for rSK2 and >300 nM for rSK3VK), suggesting that rSK3 and rSK2 can form functional heteromeric channels. To detect co-assembly of SK3 with SK1, we initially used a dominant negative construct of the human SK1 subunit (hSK1YP). hSK1YP dramatically reduced the SK3 current, supporting the idea that SK3 and SK1 subunits also interact. To determine whether these assemblies were functional we created rSK3VF, an rSK3 mutant with an enhanced affinity for tetraethylammonium chloride (TEA) (IC 50 of 0.3 mM). Co-transfection of rSK3VF and hSK1 produced currents with a sensitivity to TEA not different from that of hSK1 alone (IC 50 ϳ15 mM). 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.Small conductance Ca 2ϩ -activated potassium channels (SK 1 channels) are widely expressed throughout the central and peripheral nervous systems. In many neurons SK channels underlie some components of the post-spike after-hyperpolarization (see e.g. Ref. 1). They also have important functions in nonneuronal 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 1848) 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). In both Xenopus oocytes and mammalian cell lines, expression of the rat homologues of SK2 and SK3 (rSK2 and rSK3 respectively) results in the formation of functional homomeric SK channels. Further, both homomeric rSK2 and homomeric rSK3 channels can be blocked by either apamin or UCL 1848 at concentrations that are similar to those reported for native channels (8). The potencies of both compounds depend on the subunit composition of the channel, with the IC 50 for blocking homomeric SK2 channels being ϳ18-fold lower than for SK3.The behavior of SK1 is different to that of other SK genes....

Adenovirus DNA binding protein is a multifunctional protein essential for viral DNA replication. To investigate the role of the DNA binding protein in this process its interaction with partial DNA duplexes was examined. Duplex regions of DNA, created when a short DNA strand is annealed to its complementary sequence present in the single stranded form of M13 phage DNA, were efficiently unwound by DNA binding protein in a reaction that required neither ATP nor MgCl2. The unwinding activity of DNA binding protein was reduced by conditions which increased the stability of DNA duplexes. DNA unwinding by DNA binding protein was highly co-operative and required the single stranded DNA to be completely coated with the protein. Completely double stranded DNA could also be unwound by DNA binding protein but this reaction was sensitive to the G+C content of the DNA and could only be observed with relatively short DNA duplexes up to 45 base pairs in length. When these short double stranded DNA molecules contained binding sites for the transcription factors NFI and NFIII addition of the cognate factor blocked DNA binding protein mediated unwinding of the particular DNA duplex. Cleavage of DNA binding protein with chymotrypsin and isolation of the 39,000 molecular weight C-terminal fragment indicated that the unwinding activity was located in this domain of the protein. In support of this contention a monoclonal antibody, which had previously been mapped to this region, specifically inhibited the DNA unwinding activity. These activities of DNA binding protein are likely to be involved in DNA replication, where the destabilisation of DNA duplexes could be important both during initiation and elongation.

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Previous studies in enterocytes isolated from the villus region of small intestinal epithelium have identified a macroscopic current carried by Cl−. In this work a single-channel patch-clamp study was carried out in the same cells, and a spontaneously active, outwardly rectifying Cl− channel was identified and proposed to underlie the whole cell current. The channel had conductances of 62 and 19 pS at 80 and −80 mV, respectively, in symmetrical Cl− solutions in excised patches. Similar activity was seen in cell-attached patches, but only outward currents could be discerned in this configuration. The activity of the channel, measured as open probability, was independent of intracellular calcium levels and voltage. The selectivity sequence for different anions was SCN− > I− > Br− > Cl− > F− > (gluconate, glutamate, [Formula: see text]). The channel was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), verapamil, and 4-hydroxytamoxifen (but not by tamoxifen), with potencies similar to those observed for Cl− channels previously described in other cells. Inhibition by trinitrophenyladenosine 5′-triphosphate was also observed but only at depolarized potentials. At 50 mV the half-maximal inhibitory concentration was 18 nM. It is proposed that this channel plays a role in transepithelial Cl−transport and certain regulatory Cl− fluxes.