Separable effects of human Kvβ1.2 N- and C-termini on inactivation and expression of human Kv1.4

1. The Kv beta subunits of voltage-gated K+ channels alter the functional expression and gating of non- or slowly inactivating Kv alpha 1 subunits via two separate domains. To determine how Kv beta subunits modulate a rapidly inactivating Kv alpha 1 subunit, we did two-microelectrode voltage clamp experiments on human Kv1.4 voltage-gated K+ channels 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 Delta 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 or 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 50 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 the rate of inactivation and shifted the inactivation curve to more hyperpolarized potentials, but had no effect on deactivation. Kv beta 1-C, Kv beta 2 and Kv beta 1.2 slowed recovery from inactivation similarly, thereby excluding involvement of the beta-ball. Kv beta 1.2 produced an additional more rapid, voltage-dependent component of inactivation, significantly reduced peak outward current and shifted steady-state inactivation towards hyperpolarized potentials. 4. Yeast two-hybrid studies showed that alpha-beta interaction was restricted to the N-terminus of Kv1.4 and the C-terminus of Kv beta 1.2 or Kv beta 2. Direct interaction with the alpha-ball did not occur. Our interpretation is that 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 has three effects on Kv1.4, the first two of which it shares with Kv beta 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 allosterically. 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 present, competition for their respective binding sites slows the block produced by either ball.