SCAM analysis reveals a discrete region of the pore turret that modulates slow inactivation in Kv1.5

Abstract

In Kv1.5, protonation of histidine 463 in the S5-P linker (turret) increases the rate of depolarization-induced inactivation and decreases the peak current amplitude. In this study, we examined how amino acid substitutions that altered the physico-chemical properties of the side chain at position 463 affected slow inactivation and then used the substituted cysteine accessibility method (SCAM) to probe the turret region (E456-P468) to determine whether residue 463 was unique in its ability to modulate the macroscopic current. Substitutions at position 463 of small, neutral (H463G and H463A) or large, charged (H463R, H463K, and H463E) side groups accelerated inactivation and induced a dependency of the current amplitude on the external potassium concentration. When cysteine substitutions were made in the distal turret (T462C-P468C), modification with either the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) or negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate reagent irreversibly inhibited current. This inhibition could be antagonized either by the R487V mutation (homologous to T449V in Shaker) or by raising the external potassium concentration, suggesting that current inhibition by MTS reagents resulted from an enhancement of inactivation. These results imply that protonation of residue 463 does not modulate inactivation solely by an electrostatic interaction with residues near the pore mouth, as proposed by others, and that residue 463 is part of a group of residues within the Kv1.5 turret that can modulate P/C-type inactivation.

Interaction of propofol with voltage-gated human Kv1.5 channel through specific amino acids within the pore region

“…However, a previous study showed that the alanine substitution of His463 (H463A) in hKv1.5 channel results in a marked acceleration of current inactivation (Eduljee et al, 2007). We therefore substituted His463 to cysteine (H463C), which has been demonstrated to exhibit an inactivation rate qualitatively similar to that of the wild type hKv1.5 (Eduljee et al, 2007). It should also be noted that Thr480, Ile508 and Val516 are predicted to face toward the central cavity (Decher et al, 2006;Eldstrom et al, 2007;Tamargo et al, 2009).…”

“…Wherever possible, alanine was chosen for the substitution of wild type amino acids because it is expected to have small effects on the protein secondary structure (Eldstrom et al, 2007;Hockerman et al, 1995;Mitcheson et al, 2000). However, a previous study showed that the alanine substitution of His463 (H463A) in hKv1.5 channel results in a marked acceleration of current inactivation (Eduljee et al, 2007). We therefore substituted His463 to cysteine (H463C), which has been demonstrated to exhibit an inactivation rate qualitatively similar to that of the wild type hKv1.5 (Eduljee et al, 2007).…”

Voltage Clamp Fluorimetry Reveals a Novel Outer Pore Instability in a Mammalian Voltage-gated Potassium Channel

“…Other mutations in Shaker channels that modulate slow inactivation include E418 and V463 ( Hoshi et al, 1991 ;Larsson and Elinder, 2000 ), but unfortunately we were unable to obtain current expression or fl uorescence recordings in oocytes from the equivalent residues E456Q or E456C in Kv1.5 (see also Eduljee et al, 2007 ), or the equivalent residues to V463 in Shaker . However, another residue that modulates P/C-type inactivation in Kv1.5 is the His in the pore turret ( Kehl et al, 2002 ).…”

Identification of an Evolutionarily Conserved Extracellular Threonine Residue Critical for Surface Expression and Its Potential Coupling of Adjacent Voltage-sensing and Gating Domains in Voltage-gated Potassium Channels

“…9), in which the nonconservative mutants, with the partial exception of E502T, fail to reach the cell surface and express currents. Although Glu-502 is important for gating (42,43), it clearly plays an additional trafficking role. The possibility that these biophysical and trafficking roles may be separable would rationalize why E502T shows partial surface expression but low currents.…”

“…The possibility that these biophysical and trafficking roles may be separable would rationalize why E502T shows partial surface expression but low currents. In addition, the absent or weak currents seen in various Kv1.5 (42) and shaker B⌬ (43) channel mutants at the position equivalent to Glu-502 can now be rationalized as a failure in trafficking rather than purely an effect on channel gating (43).…”