Structure and complex transcription pattern of the mouse SK1 K(Ca) channel gene, KCNN1.

Small conductance calcium-gated K(+) channels (SK channels) are encoded by the three SK genes, SK1, SK2, and SK3. These channels likely contribute to slow synaptic afterhyperpolarizations of apamin-sensitive and apamin-insensitive types. SK channels are also widely expressed outside the nervous system. The mouse SK1 gene comprises at least 12 exons extending across 19.8 kb of genomic DNA. This gene encodes a complex pattern of alternatively spliced SK1 transcripts widely expressed among mouse tissues. These transcripts exhibit at least four distinct 5'-nucleotide sequence variants encoding at least two N-terminal amino acid sequences. Optional inclusion of exons 7 and 9, together with two alternate splice donor sites in exon 8, yields transcripts encoding eight variant C-terminal amino acid sequences for SK1. These include an altered putative S6 transmembrane span, modification of the C-terminal cytoplasmic domain binding site for calmodulin, and generation of two alternate predicted binding sites for PDZ domain-containing proteins. 20 of the 32 predicted mouse SK1 transcripts are expressed in brain at levels sufficient to allow consistent detection, and encode 16 SK1 polypeptide variants. Only four of these 16 polypeptides preserve the ability to bind calmodulin in a Ca(2+)-independent manner. Mouse SK1 also exhibits novel, strain-specific, length polymorphism of a polyglutamate repeat in the N-terminal cytoplasmic domain. The evolutionary conservation of this complex transcription pattern suggests a possible role in the tuning of SK1 channel function.

Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation.

Although K-Cl cotransporter (KCC1) mRNA is expressed in many tissues, K-Cl cotransport activity has been measured in few cell types, and detection of endogenous KCC1 polypeptide has not yet been reported. We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flanking genomic regions and mapped the mKCC1 gene to chromosome 8. Three anti-peptide antibodies raised against recombinant mKCC1 function as immunoblot and immunoprecipitation reagents. The tissue distributions of mKCC1 mRNA and protein are widespread, and mKCC1 RNA is constitutively expressed during erythroid differentiation of ES cells. KCC1 polypeptide or related antigen is present in erythrocytes of multiple species in which K-Cl cotransport activity has been documented. Erythroid KCC1 polypeptide abundance is elevated in proportion to reticulocyte counts in density-fractionated cells, in bleeding-induced reticulocytosis, in mouse models of sickle cell disease and thalassemia, and in the corresponding human disorders. mKCC1-mediated uptake of (86)Rb into Xenopus oocytes requires extracellular Cl(-), is blocked by the diuretic R(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2, 3-dihydro-1-oxo-1H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling, N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.

AE2 anion exchanger polypeptide is a homooligomer in pig gastric membranes: a chemical cross-linking study.

Although considerable information is available on the oligomeric states of the AE1 (band 3) anion exchanger, little is known about the physiological state of the polypeptides encoded by the nonerythroid AE genes, AE2 and AE3. We have previously characterized the proteolytic susceptibility of native pig gastric AE2. In the course of studies in which pig gastric membranes were treated with the AE2 transport antagonist, DIDS, we noted evidence for cross-linking of AE2 proteolytic fragments to higher-order oligomeric forms. We have characterized the ability of DIDS and of selected N-hydroxysuccinimide cross-linking agents to increase the proportion of SDS-resistant oligomers of pig gastric AE2 and its proteolytic fragments. Cross-linking exhibited time and concentration dependence. N-Terminal protein sequencing proved that DIDS treatment created AE2 homodimers. Putative homotetramers were also observed. Protomers were cross-linked via residues within the C-terminal 40 kDa of AE2. Prior proteolytic cleavage of AE2 in membranes resulted in decreased yield of subsequently cross-linked products. AE2 cross-linking could not be detected in membranes pretreated by hypotonic wash and freeze-thaw. The results are interpreted in light of the deduced amino acid sequence of the transmembrane domain of pig AE2.

cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation.

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed in Xenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.