Mammalian high conductance, calcium-activated potassium (maxi-K) channels are composed of two dissimilar subunits, alpha and beta. We have examined the functional contribution of the beta subunit to the properties of maxi-K channels expressed heterologously in Xenopus oocytes. Channels from oocytes injected with cRNAs encoding both alpha and beta subunits were much more sensitive to activation by voltage and calcium than channels composed of the alpha subunit alone, while expression levels, single-channel conductance, and ionic selectivity appeared unaffected. Channels from oocytes expressing both subunits were sensitive to DHS-I, a potent agonist of native maxi-K channels, whereas channels composed of the alpha subunit alone were insensitive. Thus, alpha and beta subunits together contribute to the functional properties of expressed maxi-K channels. Regulation of co-assembly might contribute to the functional diversity noted among members of this family of potassium channels.

We report here a characterization of two families of calcium-activated K ؉ channel ␤-subunits, ␤2 and ␤3, which are encoded by distinct genes that map to 3q26.2-27. A single ␤2 family member and four alternatively spliced variants of ␤3 were investigated. These subunits have predicted molecular masses of 27.1-31.6 kDa, share ϳ30 -44% amino acid identity with ␤1, and exhibit distinct but overlapping expression patterns. Coexpression of the ␤2 or ␤3a-c subunits with a BK ␣-subunit altered the functional properties of the current expressed by the ␣-subunit alone. The ␤2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the ␤3a-c subunits resulted in only partial inactivation of the current, and the ␤3b subunit conferred an apparent inward rectification. Furthermore, unlike the ␤1 and ␤2 subunits, none of the ␤3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these ␤-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.

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The abilities of three synthetic oligosaccharides to accelerate antithrombin inhibition of ten clotting or fibrinolytic proteinases were compared with those of unfractionated, fractionated high-affinity and low-molecular-weight heparins. The results show that the anticoagulant effects of the latter three heparins under conditions approximating physiologic are exerted almost exclusively by acceleration of the inactivation of thrombin, factor Xa and factor IXa to near diffusion-controlled rate constants of approximately 10(6) - 10(7) M(-1).s(-1). All other proteinases are inhibited with at least 20-fold lower rate constants. The anti-coagulant ability of the synthetic regular (fondaparinux) and high-affinity (idraparinux) pentasaccharides is due to a common mechanism, involving acceleration of only factor Xa inhibition to rate constants of approximately 10(6) M(-1).s(-1) . A synthetic hexadecasaccharide, containing both the pentasaccharide sequence and a proteinase binding site, exerts its anticoagulant effect by accelerating antithrombin inactivation of both thrombin and factor Xa to rate constants of approximately 10(6) - 10(7) M(-1).s(-1), although thrombin appears to be the more important target. In contrast, factor IXa inhibition is appreciably less stimulated. The conformational change of antithrombin induced both by the pentasaccharides and longer heparins contributes substantially, approximately 150-500-fold, to accelerating the inactivation of factors Xa, IXa and VIIa and moderately, approximately 50-fold, to that of factor XIIa and tissue plasminogen activator inhibition. The bridging effect due to binding of antithrombin and proteinase to the same, long heparin chain is dominating, approximately 1000-3000-fold, for thrombin inhibition and is appreciably smaller, although up to approximately 250-350-fold, for the inactivation of factors IXa and XIa. These results establish the proteinase targets of heparin derivatives currently used in or considered for thrombosis therapy and give new insights into the mechanism of heparin acceleration of antithrombin inhibition of proteinases.