Abstract
N-type inactivation of rat Kv1.4 channels with one, two, or four inactivation balls was investigated using homogeneous populations of channels expressed in Xenopus oocytes. Tandem dimeric and tetrameric constructs of Kv1.4 were made. Channels encoded by tandem cDNAs Kv1. 4-Kv1.4Delta1-145 and Kv1.4-[Kv1.4Delta1-145](3) have two or only one tethered inactivation ball, respectively, whereas Kv1.4 itself encodes channels having four inactivation balls. The time constants for inactivation of macroscopic currents were increased significantly as the number of inactivation balls was decreased, whereas the time constants for recovery from inactivation were not modified. The ratios of the rate constants of inactivation (k(inact)) of Kv1.4-Kv1.4Delta1-145 and Kv1.4-[Kv1.4Delta1-145](3) channels to that of the Kv1.4 channel were 0.65 and 0.4, respectively, whereas the ratios of the rate constant of recovery (k(rec)) of these channels to that of Kv1.4 were almost unity. The rate constants k(inact) for channels having two and four inactivation balls are smaller than those that would be expected if inactivation balls on each channel are independent, suggesting some interaction occurs between inactivation balls. Furthermore, noninactivating current became apparent as the number of inactivation balls on a channel was decreased.
Highlights
Isolation of the clone encoding the Shaker Kϩ channel from Drosophila has made it possible to examine the molecular mechanism of N-type inactivation of A-type Kϩ channels
The dimeric cDNA encodes a hybrid channel that is composed of Kv1.4 and Kv1.2 subunits with 1:1 stoichiometry
Channels formed by the partial tandem polypeptide of Kv1.4 have two tethered inactivation balls, whereas the full tandem dimeric cDNA encodes channels with two tethered inactivation balls and two positioned in the loops connecting the subunits (Fig. 1B, b and c)
Summary
Dimeric cDNA Construction—Two different dimeric cDNA constructs were generated from the cDNA clone, Kv1.4 isolated from rat cardiac muscle [23]. Fragments II or IIЈ and the MluI/NotI fragment from Kv1.4 in pBluescript were ligated into the corresponding sites of the 3Ј end-modified intermediate construct digested with SpeI and NotI. The PCR product was ligated into a partial dimeric cDNA in pBluescript digested with BstEII and EcoRI (3Ј end modified partial dimeric cDNA). The amplified fragment was ligated into the corresponding site in a partial dimeric cDNA following digestion with HindIII and MluI (5Ј end modified partial dimeric cDNA). Both 3Ј and 5Ј end modified partial dimeric cDNAs were digested by SplI and NotI and ligated into the complete tandem tetrameric constructs. Figures, and tables are given as means Ϯ S.E
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