Small conductance Ca2+-activated K+ (SK) channels have been cloned from mammalian brain, but little is known about the molecular characteristics of SK channels in nonexcitable tissues. Here, we report the isolation from rat liver of an isoform of SK3. The sequence of the rat liver isoform differs from rat brain SK3 in five amino acid residues in the NH3 terminus, where it more closely resembles human brain SK3. SK3 immunoreactivity was detectable in hepatocytes in rat liver and in HTC rat hepatoma cells. Human embryonic kidney (HEK-293) cells transfected with liver SK3 expressed 10 pS K+ channels that were Ca2+ dependent (EC(50) 630 nM) and were blocked by the SK channel inhibitor apamin (IC(50) 0.6 nM); whole cell SK3 currents inactivated at membrane potentials more positive than -40 mV. Notably, the Ca2+ dependence, apamin sensitivity, and voltage-dependent inactivation of SK3 are strikingly similar to the properties of hepatocellular and biliary epithelial SK channels evoked by metabolic stress. These observations raise the possibility that SK3 channels influence membrane K+ permeability in hepatobiliary cells during liver injury.

Cell volume recovery in response to swelling requires reorganization of the cytoskeleton and fluid efflux. We have previously shown that electrolyte and fluid efflux via K ؉ and Cl ؊ channels is controlled by swelling-induced activation of phospholipase C␥ (PLC␥). Recently, integrin engagement has been suggested to trigger responses to swelling through activation of Rho family GTPases and Src kinases. Because both PLC␥ and Rho GTPases can be regulated by Src during integrin-mediated cytoskeletal reorganization, we sought to identify swelling-induced Src effectors. Upon hypotonic challenge, Src was rapidly activated in transient plasma membrane protrusions, where it colocalized with Vav, an activator of Rho GTPases. Inhibition of Src with PP2 attenuated phosphorylation of Vav. PP2 also attenuated phosphorylation of PLC␥, and inhibited swelling-mediated activation of K ؉ and Cl ؊ channels and cell volume recovery. These findings suggest that swelling-induced Src regulates cytoskeletal dynamics, through Vav, and fluid efflux, through PLC␥, and thus can coordinate structural reorganization with fluid balance to maintain cellular integrity.

The self-splicing rRNA intron of Tetrahymena tbermopbila contains two stem-loop structures (P9.1 and P9.2) near its 3' end that are not conserved among group I introns. As a step toward deriving the smallest active selfsplicing RNA, 78 nucleotides encompassing P9.1 and P9.2 have been deleted. This deletion has no effect on the core catalytic activity of the intron, as judged by its ability to catalyze poly{C) polymerization and other related reactions. In contrast, reactions at the 3' splice site of the rRNA precursor-exon ligation and intermolecular exon ligation-take place with reduced efficiency, and exon ligation becomes rate-limiting for self-splicing. Moreover, intermolecular exon ligation with pentaribocytidylic acid is inaccurate, occurring primarily at a cryptic site in the 3' exon. A deletion of 79 nucleotides that disrupts P9, as well as removing P9.1 and P9.2, has more severe effects on both the first and second steps of splicing. P9, a conserved helix at the 5' edge of the deletion point, can form stable alternative structures in the deletion mutants. This aberrant folding may be responsible for the reduced activity and accuracy of reactions with mutant precursors. Analysis of the cryptic site suggests that choice of the 3' splice site may not only depend on sequence but also on proximity to P9. In the course of these studies, evidence has been obtained for an altemative 5' exon-binding site distinct from the normal site in the internal guide sequence. Many RNA transcripts contain domains called introns (intervening sequences, IVSs) that are not found in the mature RNA but are removed by a process called RNA splicing. Some introns are capable of splicing in the ab sence of proteins or other macromolecular factors. Therefore, the information required for this self-splicing is contained within the native structure of the intron itself (Kruger et al. 1982).Group I introns splice by a two-step transesterification mechanism that is initiated by nucleophilic attack of a guanosine cofactor (Cech 1987). The 60 reported members of this class are heterogeneous with respect to length, ranging from 258 to 2641 nucleotides (Michel and Cummings 1985;Trinkl and Wolf 1986), and show little primary sequence homology. However, at the sec ondary structure level, there is remarkable conservation.

MLCK resolves membrane blebbing induced by osmotic cell swelling. Cell swelling also stimulates the formation of a Src–MLCK complex, which with cortactin and dynamin forms actin-based structures at the base of the cell to facilitate membrane retrieval for volume recovery.

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