C‐Type Inactivation Involves a Significant Decrease in the Intracellular Aqueous Pore Volume of Kv1.4 K⁺ Channels Expressed in Xenopus Oocytes

Abstract

Channels are water‐filled membrane‐spanning proteins, which undergo conformational changes as they gate, i.e. open or close. These conformational changes affect both the shape of the channel and the volume of the water‐filled pore. We measured the changes in pore volume associated with activation, deactivation, C‐type inactivation and recovery in an N‐terminal‐deleted mutant of the Kv1.4 K+ channel (Kv1.4ΔN) expressed in Xenopus oocytes. We used giant‐patch and cut‐open oocyte voltage clamp techniques and applied solutes which are too large to enter the pore mouth to exert osmotic pressure and thus favour smaller pore volume conformations. Applied intracellular osmotic pressure (300 mm sucrose) sped inactivation (time constants (τinactivation): control, 0.66 ± 0.09 s; hyperosmotic solution, 0.29 ± 0.04 s; n= 5, P < 0.01), sped deactivation (τdeactivation: control, 18.8 ± 0.94 ms; hyperosmotic solution, 8.01 ± 1.92 ms; n= 5, P < 0.01), and slowed activation (τactivation: control, 1.04 ± 0.05 ms; hyperosmotic solution, 1.96 ± 0.31 ms; n= 5, P < 0.01). These effects were reversible and solute independent. We estimated the pore volume change on inactivation to be about 4500 Å3. Osmotic pressure had no effect when applied extracellularly. These data suggest that the intracellular side of the pore closes during C‐type inactivation and the volume change is similar to that associated with activation or deactivation. This is also similar to the pore volume estimated from the crystal structure of KcsA and MthK K+ channels. Intracellular osmotic pressure also strongly inhibited re‐opening currents associated with recovery from inactivation, which is consistent with a physical similarity between the C‐type inactivated and resting closed state.

Shab K+channel slow inactivation

“…17-21) as well as locations along the S6 segment (e.g., refs. 3,[22][23][24][25], in particular the central cavity of the pore. Regarding the latter, and of particular interest to this study, is the recent demonstration that internal TEA (TEA i ) inhibits the slow inactivation of Kv2.1 and Shaker channels.…”

Closed-state inactivation in Kv4.3 isoforms is differentially modulated by protein kinase C

“…In contrast, other data suggest that the fast component of inactivation is due to occlusion of the inner vestibule by the NH 2 terminus (20). Furthermore, the slower components of inactivation in Kv4 differ from C-type inactivation reported for Shaker K ϩ channels, where pore closure plays a critical role (7,17,19,23,25,40,44,52). In contrast to Shaker K ϩ channels, there is a substantial degree of inactivation in the Kv4 family that occurs from closed states (5,6,9,17,49).…”

“…In contrast to the rest of the channel, there is very little structural information about the COOH-terminal cytoplasmic region of rapidly inactivating K ϩ channels. What little is known has been derived from electron microscopic analysis of Kv4.2 (23) and suggests that the phosphorylation sites lie at the periphery of the channel complex and are unlikely to come in contact. If this is true, then each phosphorylated threonine at position 504 should be able to contribute to the enhancement of closed-state inactivation, regardless of the phosphorylation state of the other three.…”

“…4. In these channels, the rapid and slow components of inactivation result from a ball and chain mechanism occluding the inner vestibule (N-type), and pore closure (C-type) (7,12,19,23,25,30,40,41,46,52). In contrast, Kv4 inactivation is more complex; deletion of either the amphipathic region of the NH 2 terminus of Kv4.1 or a largely hydrophilic region in the COOH terminus abolishes the rapid component of inactivation (20).…”

The Effects of 4-Hydroxybenzoic Acid Identified from Bamboo (Dendrocalamus asper) Shoots on Kv1.4 Channel

“…The "ball and chain" permeation mechanism of N-type inactivation occurs by blocking the pore by the "ball" preventing the movement of K + (46). Rapid N-type inactivation is important in developing the C-type, which is hypothesised to involve the selectivity filter, conformational changes, mutations of the pore, quinidine binding (39), intracellular pore closure (47), and osmotic pressure (48). An increase in extracellular K + concentration enhances the activation of the Kv1.4 channel by binding to the selectivity filter.…”