Separable effects of human Kvbeta1.2 N- and C-termini on inactivation and expression of human Kv1.4.
1. The Kvbeta subunits of voltage-gated K+ channels alter the functional expression and gating of non- or slowly inactivating Kvalpha1 subunits via two separate domains. To determine how Kvbeta subunits modulate a rapidly inactivating Kvalpha1 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. 4DeltaN2-146). Kv1.4 or Kv1.4DeltaN2-146 were co-expressed with either rat Kvbeta2 or human Kvbeta1.2. To separate domain effects, we also used a mutant of Kvbeta1.2 lacking the unique 79 amino acid N-terminal beta-ball domain (Kvbeta1-C). 2. For the mutant Kv1.4DeltaN2-146 we found that Kvbeta1-C or Kvbeta2 increased current amplitude without altering activation or inactivation. By contrast Kvbeta1.2 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, Kvbeta1-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. Kvbeta1-C, Kvbeta2 and Kvbeta1.2 slowed recovery from inactivation similarly, thereby excluding involvement of the beta-ball. Kvbeta1.2 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 Kvbeta1. 2 or Kvbeta2. Direct interaction with the alpha-ball did not occur. Our interpretation is that Kvbeta1-C and Kvbeta2 enhanced N-type inactivation produced by the Kv1.4 alpha-ball allosterically. 5. We propose that Kvbeta1.2 has three effects on Kv1.4, the first two of which it shares with Kvbeta2. First, Kvbeta1-C and Kvbeta2 have a current-enhancing effect. Second, Kvbeta1-C and Kvbeta2 increase block by the alpha-ball allosterically. Third, the beta-ball of Kbeta1.2 directly blocks both Kv1.4 and Kv1.4DeltaN2-146. When both alpha- and beta-balls are present, competition for their respective binding sites slows the block produced by either ball.
Accili
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Rammelkamp Center for Education and Research, MetroHealth Campus, 2500 MetroHealth Drive, Cleveland, OH 44109-1998, USA.
Kuryshev
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Wible
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Brown
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D000818:Animals; D001711:Biotransformation / Q000187:drug effects / Q000502:physiology; D004558:Electric Stimulation; D004594:Electrophysiology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D051663:Kv1.2 Potassium Channel; D051665:Kv1.4 Potassium Channel; D008564:Membrane Potentials / Q000502:physiology; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D014981:Xenopus; D015003:Yeasts / Q000378:metabolism
C496969:KCNA2 protein, human; C496977:KCNA4 protein, human; D051663:Kv1.2 Potassium Channel; D051665:Kv1.4 Potassium Channel; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated