Modification of C-type inactivatingShaker potassium channels by chloramine-T

Abstract

Shaker potassium channels undergo a slow C-type inactivation which can be hastened dramatically by single-point mutations in or near the pore region. We found that the oxidizing agent chloramine-T (Chl-T) causes an irreversible loss of current for those mutants which show C-type inactivation. For several mutants at position T449, which show a wide spectrum of inactivation time constants, the time constant of current rundown induced by Chl-T correlated with the speed of inactivation. Rundown was accelerated when the channels were in the inactivated state but rundown also occurred when channels were not opened or inactivated. Apparently, only those channels which can undergo C-type inactivation are accessible to Chl-T. In order to gain information about the target amino-acid residue for the action of Chl-T and the structural rearrangements occurring during C-type inactivation, several mutant channel proteins were compared with respect to their response to Chl-T. Since Chl-T can oxidize cysteine and methionine residues, we mutated the possible targets in and close to the pore region, namely C462 to A, and M440 and M448 to I. While the residues M440 and C462 were not important for channel rundown, mutation of M448 to I made the channels more resistant to Chl-T by about one order of magnitude. While inactivation was accelerated upon application of Chl-T in most mutants, mutation of M448 to I abolished this effect on the time course of inactivation, indicating that M448 is one of the target residues for Chl-T.

Modulation of potassium channel function by methionine oxidation and reduction

“…At greater concentrations (1 mM), however, Ch-T slowed the apparent activation time course and reduced the peak amplitudes of the ShC͞B currents. These effects of Ch-T at the high concentrations are probably mediated by the effects on other gating processes (27). Internal DTT (5 mM) in the inside-out patches did not have any consistent effects on the ShC͞B currents (data not shown).…”

C-type inactivation of voltage-gated K+ channels: Pore constriction or dilation?

“…The crystal structure of the K v 1.2-2.1 chimera shows that these four residues (V377/ T449 ) are 15 Å apart, and it seems unlikely that the proximity required for multiple binding of Cd 2+ , ∼4 Å (Maher et al, 2004), can be achieved without a very large (and, we think, somewhat improbable) rearrangement. Although ample evidence shows that changes in the reactivity of engineered cysteines at the external mouth of the pore accompany C-type inactivation in K V channels (Yellen et al, 1994; Liu et al, 1996; Schlief et al, 1996; Larsson and Elinder, 2000), there is no direct proof that these changes depend on pore mouth constriction.…”

Modulation of slow inactivation in human cardiac Kv1.5 channels by extra‐ and intracellular permeant cations

“…In many Shaker channels, mutations of T449 or the equivalent site to Y or V increases the time constant for C_type inactivation to > 10 s (Lopez-Barneo et al 1993), whereas the positively charged residue K gives a rapid C_type inactivation (Schlief et al 1996). Kv1.5 shows an intermediate rate of pore-dependent inactivation with R487 at the equivalent site, and is only mildly sensitive to extracellular TEA at concentrations up to 50 mÒ (Fedida et al 1993;Snyders et al 1993;Grissmer et al 1994).…”