Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels.

Abstract

Voltage-activated K+ channels are a family of closely related membrane proteins that differ in their gating behavior, conductance, and pharmacology. A prominent and physiologically important difference among K+ channels is their rate of inactivation. Inactivation rates range from milliseconds to seconds, and K+ channels with different inactivation properties have very different effects on signal integration and repetitive firing properties of neurons. The cloned Shaker B (H4) potassium channel is an example of a K+ channel that inactivates in a few milliseconds. Recent experiments have shown that removal of an N-terminal region of the Shaker protein by site-directed deletion practically abolishes this fast inactivation, but the modified channel does still inactivate during a prolonged depolarization lasting many seconds. Here we report that this remnant inactivation must occur by a distinct mechanism from the rapid inactivation ofthe wild-type Shaker channel. Like the inactivation of another K+ channel [Grissmer, S. & Calahan, M. (1989) Biophys. J. 55, 203-206], this slow inactivation is retarded by the application ofa channel blocker, tetraethylammonium, to the extracellular side of the channel. By contrast, the fast inactivation of the wild-type Shaker channel is sensitive only to intracellular application of tetraethylammonium. Intracellular tetraethylammonium slows down the fast inactivation process, as though it competes with the binding of the inactivation particle.Recent experiments on the rapidly inactivated Shaker K+ channel suggest that the N-terminal part of this protein may act as a blocking particle that blocks current through the channel and thus produces fast inactivation (1, 2). The model of a blocking particle tethered to the rest of the channel (picturesquely termed the "ball-and-chain" model) was proposed many years ago to explain the properties of inactivation of voltage-activated Na' channels (3, 4). Inactivation of both Na' and Shaker K+ channels can be removed by mild protease treatment of the intracellular side of the membrane (1, 5, 6). Inactivation of both types of channels can also be mimicked by the application of intracellular compounds that block current through the channel after the activation gates have opened (7)(8)(9)(10)(11)(12)(13). One such compound is a soluble 20-amino acid peptide with the sequence ofthe N terminus ofthe Shaker protein, which mimics the fast inactivation of this channel when applied to the intracellular side of a Shaker channel from which a region near the N terminus has been genetically removed (14). These results suggest that the N terminus of the Shaker protein is a strong candidate for the tethered "ball" and that the inactivation process is localized to the intracellular portion of the channel.These results on the rapid inactivation of Shaker stand in contrast to studies of the much slower inactivation process seen in a delayed-rectifier K+ channel in lymphocytes. This inactivation process, which occurs over a period of hundreds of millisecond...

Molecular Basis for the Inactivation of Ca2+- and Voltage-Dependent BK Channels in Adrenal Chromaffin Cells and Rat Insulinoma Tumor Cells

“…Although inactivation of BK i channels shares some features in common with N-terminal-mediated inactivation (Solaro et al, 1997), there are also clear differences. Unlike inactivation mediated by the N terminal of the Shak erB ␣-subunit (Choi et al, 1991;Demo and Yellen, 1991), inactivation mediated by the tethered ␤3 N-terminal domain involves a binding site not influenced by occupancy of the ion permeation pathway by QX-314. Thus, the ␤3 N-terminal domain appears to block the BK channel at a position probably not homologous with the Shaker pore site acted on by the Shak erB N-terminal structures.…”

“…Inactivation of native BK i channels also exhibits one feature that distinguishes it from inactivation of the Shaker family of voltage-dependent K ϩ channels (Choi et al, 1991) and from inactivation of BK channels observed in hippocampal neurons (Hicks and Marrion, 1998). Specifically, cytosolic blockers of BK channels do not slow the native BK i inactivation process (Solaro et al, 1997), indicative that occupancy of a site within the mouth of the ion permeation pathway does not hinder movement of the inactivation domains to their blocking sites.…”

“…In Figure 5A, the cytosolic application of 2 mM QX-314 reduced the averaged peak current to ϳ24% of the control amplitude, having only small effects on i . For a simple model in which QX-314 is proposed to compete with the native inactivation process, a reduction of current to 24% is predicted to produce a 4.17-fold prolongation of i (Choi et al, 1991;Ding et al, 1998). For four patches, 2 mM QX-314 produced a 1.18 Ϯ 0.07-fold prolongation of i .…”

“…As with the ShakerB channel (Hoshi et al, 1990;Gomez-Lagunas and Armstrong, 1995), BK i inactivation arises from multiple trypsin-sensitive cytosolic domains (Ding et al, 1998). However, in contrast to the open channel-like blockade produced by the ShakerB N-terminal peptide (Choi et al, 1991;Demo and Yellen, 1991), cytosolic blockers of the BK channel pore do not compete with the native inactivation domain (Solaro et al, 1997). Previous work has failed to reveal a Slo ␣-subunit splice variant that might account for the inactivating phenotype (Saito et al, 1997).…”

On the activation—inactivation coupling in Shaker potassium channels

“…First, internal TEA blocks only open channels, not closed channels [91. Second, internal TEA may compete with the inactivation ball for the same site or two slightly different sites [10]. As the inactivation ball or TEA approaches W435s from the cytoplasmic side, its positive field may hinder the return of the transferring electron upon repolarization, resulting in "charge immobilization" [!…”