SEPARABLE EFFECTS OF HUMAN KV-BETA-1.2 N-TERMINI AND C-TERMINI ON INACTIVATION AND EXPRESSION OF HUMAN KV1.4

1. The Kv beta subunits of voltage-gated K+ channels alter the functio nal expression and gating of non- or slowly inactivating Kv alpha 1 su bunits via two separate domains. To determine how Kv beta subunits mod ulate a rapidly inactivating Kv alpha 1 subunit, we did two-microelect rode voltage clamp experiments on human Kv1.4 voltage-gated K+ channel s expressed heterologously in Xenopus oocytes. In addition we tested a slowly inactivating mutant of Kv1.4 lacking amino acids 2-146 of the N-terminal alpha-ball domain (Kv1.4 Delta N2-146). Kv1.4 or Kv1.4 Delt a N2-146 were co-expressed with either rat Kv beta 2 or human Kv beta 1.2. To separate domain effects, we also used a mutant of Kv beta 1.2 lacking the unique 79 amino acid N-terminal beta-ball domain (Kv beta 1-C). 2. For the mutant Kv1.4 Delta N2-146 we found that Kv beta 1-C o r Kv beta 2 increased current amplitude without altering activation or inactivation. By contrast Kv beta 1.2 produced rapid inactivation and slowed deactivation due to block produced by the beta-ball. The beta- ball also increased the rate of C-type inactivation in 5 mM, but not 5 0 mM, external K+ consistent with an effect of blockade on K+ efflux. 3. For Kv1.4, Kv beta 1-C produced a voltage-independent increase in t he rate of inactivation and shifted the inactivation curve to more hyp erpolarized potentials, but had no effect on deactivation. Kv beta 1-C , Kv beta 2 and Kv beta 1.2 slowed recovery from inactivation similarl y, thereby excluding involvement of the beta-ball. Kv beta 1.2 produce d an additional more rapid, voltage-dependent component of inactivatio n, significantly reduced peak outward current and shifted steady-state inactivation towards hyperpolarized potentials. 4. Yeast two-hybrid s tudies showed that alpha-beta interaction was restricted to the N-term inus of Kv1.4 and the C-terminus of Kv beta 1.2 or Kv beta 2. Direct i nteraction with the alpha-ball did not occur. Our interpretation is th at Kv beta 1-C and Kv beta 2 enhanced N-type inactivation produced by the Kv1.4 alpha-ball allosterically. 5. We propose that Kv beta 1.2 ha s three effects on Kv1.4, the first two of which it shares with Kv bet a 2. First, Kv beta 1-C and Kv beta 2 have a current-enhancing effect. Second, Kv beta 1-C and Kv beta 2 increase block by the alpha-ball al losterically. Third, the beta-ball of K beta 1.2 directly blocks both Kv1.4 and Kv1.4 Delta N2-146. When both alpha- and beta-balls are pres ent, competition for their respective binding sites slows the block pr oduced by either ball.