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....

To elucidate the mechanisms underlying peripheral neuropathic pain in the context of HIV infection and antiretroviral therapy, we measured gene expression in dorsal root ganglia (DRG) of rats subjected to systemic treatment with the anti-retroviral agent, ddC (Zalcitabine) and concomitant delivery of HIV-gp120 to the rat sciatic nerve. L4 and L5 DRGs were collected at day 14 (time of peak behavioural change) and changes in gene expression were measured using Affymetrix whole genome rat arrays. Conventional analysis of this data set and Gene Set Enrichment Analysis (GSEA) was performed to discover biological processes altered in this model. Transcripts associated with G protein coupled receptor signalling and cell adhesion were enriched in the treated animals, while ribosomal proteins and proteasome pathways were associated with gene down-regulation. To identify genes that are directly relevant to neuropathic mechanical hypersensitivity, as opposed to epiphenomena associated with other aspects of the response to a sciatic nerve lesion, we compared the gp120 + ddC-evoked gene expression with that observed in a model of traumatic neuropathic pain (L5 spinal nerve transection), where hypersensitivity to a static mechanical stimulus is also observed. We identified 39 genes/expressed sequence tags that are differentially expressed in the same direction in both models. Most of these have not previously been implicated in mechanical hypersensitivity and may represent novel targets for therapeutic intervention. As an external control, the RNA expression of three genes was examined by RT-PCR, while the protein levels of two were studied using western blot analysis.

1. Our aim was to identify the small-conductance Ca 2+ -activated K + channel(s) (SK) underlying the apamin-sensitive afterhyperpolarization (AHP) in rat superior cervical ganglion (SCG) neurones.2. Degenerate oligonucleotide primers designed to the putative calmodulin-binding domain conserved in all mammalian SK channel sequences were employed to detect SK DNA in a cDNA library from rat SCG. Only a single band, corresponding to a fragment of the rSK3 gene, was amplified.3. Northern blot analysis employing a PCR-generated rSK3 fragment showed the presence of mRNA coding for SK3 in SCG as well in other rat peripheral tissues including adrenal gland and liver.4. The same rSK3 fragment enabled the isolation of a full-length rSK3 cDNA from the library. Its sequence was closely similar to, but not identical with, that of the previously reported rSK3 gene.5. Expression of the rSK3 gene in mammalian cell lines (CHO, HEK cells) caused the appearance of a K + conductance with SK channel properties.6. The application of selective SK blocking agents (including apamin, scyllatoxin and newer nonpeptidic compounds) showed these homomeric SK3 channels to have essentially the same pharmacological characteristics as the SCG afterhyperpolarization, but to differ from those of homomeric SK1 and SK2 channels.7. Immunohistochemistry using a rSK3 antipeptide antibody revealed the presence of SK3 protein in the cell bodies and processes of cultured SCG neurones.8. Taken together, these results identify SK3 as a major component of the SK channels responsible for the afterhyperpolarization of cultured rat SCG neurones.

Rheumatoid arthritis (RA) is found more often in subjects carrying the HLA-DR1 antigen and some subtypes of the antigen than in those without these antigens. Analysis of probes specific for HLA-DR4 has shown that amino acids encoding positions 69-74 (EQRRAA) of the f6 chain indicates susceptibility to RA. A hexamer sequence of proteus haemolysin spanning residues 32-37 (ESRRAL) has been identified which resembles biochemically, and discriminates by charge, between HLA types associated with RA (DRi, Dw4, Dw14, Dwl5), and those not linked with the disease (DwlO, Dw13). (Ann Rheum Dis 1992; 51: 1245-1246 The association between rheumatoid arthritis (RA) and the HLA-DR4 antigen is well established, not only in white subjects but also in many other ethnic groups. HLA-DR4 can be subdivided into several subtypes, only some of which are associated with RA. In white subjects DR4/Dw4 and DR4/Dwl4 subtypes are associated with RA, whereas in Japanese subjects it is DR4/Dw15 that is the susceptibility factor. In Israeli Jews, where DR4/DwlO predominates, an association with HLA-DRI has been reported. 1 2 Analysis with synthetic oligonucleotides has shown that a particular sequence from positions 70-74 encoding the amino acids Gln-Arg-ArgAla-Ala (QRRAA), specific for DRI, Dw14, and Dw 15, showed a strong association with RA compared with controls, although another probe specific for DwlO showed no such association.