Coupling of voltage-dependent potassium channel inactivation and oxidoreductase active site of Kvbeta subunits.

The accessory beta subunits of voltage-dependent potassium (Kv) channels form tetramers arranged with 4-fold rotational symmetry like the membrane-integral and pore-forming alpha subunits (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell. 90, 943-952). The crystal structure of the Kvbeta2 subunit shows that Kvbeta subunits are oxidoreductase enzymes containing an active site composed of conserved catalytic residues, a nicotinamide (NADPH)-cofactor, and a substrate binding site. Also, Kvbeta subunits with an N-terminal inactivating domain like Kvbeta1.1 (Rettig, J., Heinemann, S. H., Wunder, F., Lorra, C., Parcej, D. N., Dolly, O., and Pongs, O. (1994) Nature 369, 289-294) and Kvbeta3.1 (Heinemann, S. H., Rettig, J., Graack, H. R., and Pongs, O. (1996) J. Physiol. (Lond.) 493, 625-633) confer rapid N-type inactivation to otherwise non-inactivating channels. Here we show by a combination of structural modeling and electrophysiological characterization of structure-based mutations that changes in Kvbeta oxidoreductase activity may markedly influence the gating mode of Kv channels. Amino acid substitutions of the putative catalytic residues in the Kvbeta1.1 oxidoreductase active site attenuate the inactivating activity of Kvbeta1.1 in Xenopus oocytes. Conversely, mutating the substrate binding domain and/or the cofactor binding domain rescues the failure of Kvbeta3.1 to confer rapid inactivation to Kv1.5 channels in Xenopus oocytes. We propose that Kvbeta oxidoreductase activity couples Kv channel inactivation to cellular redox regulation.

Mice with disrupted BK channel beta1 subunit gene feature abnormal Ca(2+) spark/STOC coupling and elevated blood pressure.

Large-conductance potassium (BK) channels in vascular smooth muscle cells (VSMCs) sense both changes in membrane potential and in intracellular Ca(2+) concentration. BK channels may serve as negative feedback regulators of vascular tone by linking membrane depolarization and local increases in intracellular Ca(2+) concentration (Ca(2+) sparks) to repolarizing spontaneous transient outward K(+) currents (STOCs). BK channels are composed of channel-forming BKalpha and auxiliary BKbeta1 subunits, which confer to BK channels an increased sensitivity for changes in membrane potential and Ca(2+). To assess the in vivo functions of this ss subunit, mice with a disrupted BKbeta1 gene were generated. Cerebral artery VSMCs from BKbeta1 -/- mice generated Ca(2+) sparks of normal amplitude and frequency, but STOC frequencies were largely reduced at physiological membrane potentials. Our results indicate that BKbeta1 -/- mice have an abnormal Ca(2+) spark/STOC coupling that is shifted to more depolarized potentials. Thoracic aortic rings from BKbeta1 -/- mice responded to agonist and elevated KCl with a increased contractility. BKbeta1 -/- mice had higher systemic blood pressure than BKbeta1 +/+ mice but responded normally to alpha(1)-adrenergic vasoconstriction and nitric oxide-mediated vasodilation. We propose that the elevated blood pressure in BKbeta1 -/- mice serves to normalize Ca(2+) spark/STOC coupling for regulating myogenic tone. The full text of this article is available at http://www.circresaha.org.

hKCNMB3 and hKCNMB4, cloning and characterization of two members of the large-conductance calcium-activated potassium channel beta subunit family.

We cloned two beta subunits of large-conductance calcium-activated potassium (BK) channels, hKCNMB3 (BKbeta1) and hKCNMB4 (BKbeta4). Profiling mRNA expression showed that hKCNMB3 expression is enriched in testis and hKCNMB4 expression is very prominent in brain. We coexpressed BK channel alpha (BKalpha) and BKbeta4 subunits in vitro in CHO cells. We compared BKalpha/beta4 mediated currents with those of smooth muscle BKalpha/beta1 channels. BKbeta4 slowed activation kinetics more significantly, led to a steeper apparent calcium sensitivity, and shifted the voltage range of BK current activation to more negative potentials than BKbeta1. BKalpha/beta4 channels were not blocked by 100 nM charybdotoxin or iberiotoxin, and were activated by 17beta-estradiol.

Gene structures and expression profiles of three human KCND (Kv4) potassium channels mediating A-type currents I(TO) and I(SA).

The four known members of the KCND/Kv4 channel family encode voltage-gated potassium channels. Recent studies provide evidence that members of the Kv4 channel family are responsible for native, rapidly inactivating (A-type) currents described in heart (I(TO)) and neurons (I(SA)). In this study, we cloned the human KCND1 cDNA, localized the KCND1 gene to chromosome Xp11.23-p11.3, and determined the genomic structure and tissue-specific expression of the KCND1, KCND2, and KCND3 genes, respectively. The open reading frame of Kv4. 1 is 1941 nucleotides long, predicting a protein of 647 amino acids. The deduced protein sequence of Kv4.1 shows an overall identity of 60% with Kv4.2 and Kv4.3L and corresponds to the common structure of voltage-gated potassium channels. KCND1-specific transcripts were detectable in human brain, heart, liver, kidney, thyroid gland, and pancreas, as revealed by Northern blot and RT-PCR experiments. The comparison of the expression patterns of the known Kv4 family members shows subtype specificity with significant overlaps. The KCND gene structures exhibit an evolutionarily conserved exon pattern with a large first exon containing the intracellular N-terminus and the putative membrane-spanning regions S1 to S5, as well as part of the pore region. The KCND3 gene contains an additional exon of 57 bp, which is not present in the other two KCND genes and gives rise to the C-terminal splice KCND3L variant with an insertion of 19 amino acids.

The anti-Dextran response of scid mice transgenic for the heavy chain of a Dextran specific IgM antibody.

Mice homozygous for the scid mutation bear a severe defect in their ability to rearrange V(D)J gene segments to yield active genes for immunoglobulin and T cell receptor molecules. In older animals few clones of B and T cells can arise at random, a phenomenon called leakyness of the scid mutation. We established scid mice carrying as a transgene the rearranged heavy chain of the IgM/lambda1 antibody MOPC 104E with specificity for the alpha(1,3) glucosidic linkages in Dextran. Despite the scid defect one-third of these mice immunized with the thymus independent antigen Dextran at 2 weeks of age, and all of those immunized at 6 weeks responded with anti-Dextran antibodies bearing the lambda light chain. This indicates that despite the scid mutation these animals had at least once successfully rearranged their endogenous lambda1 light chain gene segments and harbor Dextran specific B cells. These mice thus provided for the first time the opportunity to study the immune response of B cells of a single specificity in an environment that should, as we shall argue, be devoid of regulatory B and T cells able to recognize the idiotype of the responding cells. One week after immunization the anti-Dextran response of 5- to 6-week-old mu-transgenic scid mice amounted to 30% of the response of mu-transgenic non-scid mice but in essence both responses followed the same kinetics, reaching antibody concentrations indistinguishable from each other 8 weeks after a single dose of Dextran. Furthermore, the ready response of young mu-transgenic scid mice to this antigen by employment of endogenously rearranged lambda1 light chains allowed experiments to be done to compare the frequency of lambda1 light chain rearrangements in mu-transgenic scid mice to that in mu-transgenic non-scid mice. This was done in limiting dilution assays counting B cell precursors responsive to mitogen and differentiating in vitro to produce antibodies toward Dextran. Specific precursors were reduced to about 1% in the spleen of mu-transgenic scid mice when compared to the spleen of mu-transgenic non-scid mice; those in the peritoneal cavity lymphocyte population were reduced to about 12%.

Two genetically separable steps in the differentiation of thymic epithelium.

The development of the thymus depends initially on epithelial-mesenchymal and subsequently on reciprocal lympho-stromal interactions. The genetic steps governing development and differentiation of the thymic microenvironment are unknown. With the use of a targeted disruption of the whn gene, which recapitulates the phenotype of the athymic nude mouse, the WHN transcription factor was shown to be the product of the nude locus. Formation of the thymic epithelial primordium before the entry of lymphocyte progenitors did not require the activity of WHN. However, subsequent differentiation of primitive precursor cells into subcapsular, cortical, and medullary epithelial cells of the postnatal thymus did depend on activity of the whn gene. These results define the first genetically separable steps during thymic epithelial differentiation.