Inactivation of the cloned potassium channel mouse Kv1.1 by the human Kv3.4 ‘ball’ peptide and its chemical modification.

Abstract

1. This study used the whole-cell patch clamp technique to investigate the action of a 28-mer 'inactivation peptide' based on part of the N-terminal sequence of the human Kv3.4 K+ channel (hKv3.4 peptide) on the cloned mouse brain K+ channel mKv1.1 expressed in Chinese hamster ovary (CHO) cells, and compared this with the inactivation produced by Shaker B inactivation peptide (ShB peptide). 2. Inclusion of the hKv3.4 peptide in the patch electrode (320 /M) transformed noninactivating mKv1.1 into a rapidly inactivating current. The voltage dependence of time constants of decay and steady-state inactivation induced by hKv3.4 peptide were characteristic of an 'A-type' K+ current. 3. The hKv3.4 peptide had no effect on the voltage dependence of activation of mKvl .1, with a mid-point of activation of -8 mV, and a slope factor of 15 mV. Steady-state inactivation curves had a mid-point of inactivation of -36 mV and a slope factor of -7 mV; the time constant of recovery from inactivation at -90 mV was 1-3 s.4. The chemical modification reagents N-bromoacetamide (NBA, 100 /tM) and chloramine-T (CL-T, 500 1uM) had no effect on the fast inactivation of mKvl.1 induced by ShB peptide. In contrast, the inactivation caused by hKv3.4 peptide was removed by brief exposure to NBA and CL-T. 5. Chemical modification resulted in a hyperpolarizing shift of -8 mV (CL-T) and -11 mV (NBA) in the voltage dependence of activation of mKvl .1 in the presence of hKv3.4 peptide. 6. Chemical modification was critically dependent on the presence of a cysteine residue at position 6, and not postion 24, of hKv3.4 peptide. 7. NBA and CL-T caused only a slight inhibition of unmodified mKvl.1 current with no significant effect on the voltage dependence of mKvl .1 activation, and also had no effect on channel deactivation at -90 mV. 8. Chemical modification experiments were consistent with a selective action on the hKv3.4 peptide itself, specifically at the cysteine residue at position 6.

Sodium Channel Inactivation: Molecular Determinants and Modulation

“…CT is commonly thought to act by modifying methionine groups (479), but this explanation has been questioned (356). Besides, the action of CT cannot be highly specific as it also delays the inactivation of some types of potassium channels (377,424). CT is preferably applied for only a few minutes, since longer treatments destroy the preparations with the possible exception of squid axons (480).…”

Phosphorylation and protonation of neighboring MiRP2 sites: function and pathophysiology of MiRP2‐Kv3.4 potassium channels in periodic paralysis

“…A key role for PKC in Kv3.4 is already known; this pore-forming ␣ subunit has an amino-terminal inactivation domain of ϳ28 amino acids containing two PKC sites where phosphorylation acts to alter secondary structure and impair inactivation (22)(23)(24)(25). Thus, MiRP2-Kv3.4 channels can be regulated by action of PKC on both MiRP2 and Kv3.4 subunits, with phosphorylation leading to increased current due to activation at more negative potentials and reduced inactivation.…”

Structural determinants of Kvβ1.3-induced channel inactivation: a hairpin modulated by PIP2

“…For example, deletion of 10 amino acids from the N terminus of Kvβ1.3 (Uebele et al , 1998) causes a loss of function as does the L7E mutation in Shaker B α‐subunits (Hoshi et al , 1990). Cysteine residues at position 7 of Kvβ1.1 (Rettig et al , 1994), position 6 of Kv3.4 (Stephens and Robertson, 1995) or position 13 of Kv1.4 (Ruppersberg et al , 1991) confer a redox sensitivity to channel inactivation. The loss of function by L7E or L7R in Shaker B (Hoshi et al , 1990) can be mimicked by phosphorylation of Y8 that prevents formation of a functional hairpin structure (Encinar et al , 2002).…”