Chloride concentration affects Kv channel voltage-gating kinetics: Importance of experimental anion concentrations

Abstract

Chloride concentration has been shown to have a dramatic impact on protein folding and subsequent tertiary conformation [K.D. Collins, Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process, Methods 34 (2004) 300–311; I. Jelesarov, E. Durr, R.M. Thomas, H.R. Bosshard, Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper), Biochemistry 37 (1998) 7539–7550]. As it is known that Kv channel gating is linked to the stability of the cytoplasmic T1 multimerization domain conformation [D.L. Minor, Y.F. Lin, B.C. Mobley, A. Avelar, Y.N. Jan, L.Y. Jan, J.M. Berger, The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel, Cell 102 (2000) 657–670; B.A. Yi, D.L. Minor Jr., Y.F. Lin, Y.N. Jan, L.Y. Jan, Controlling potassium channel activities: interplay between the membrane and intracellular factors, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 11016–11023] and that intracellular chloride concentration has been linked to Kv channel kinetics [L.K. Bekar, W. Walz, Intracellular chloride modulates A-type potassium currents in astrocytes, Glia 39 (2002) 207–216; W.B. Thoreson, S.L. Stella, Anion modulation of calcium current voltage dependence and amplitude in salamander rods, Biochim. Biophys. Acta 1464 (2000) 142–150], the objective of the present study was to address how chloride concentration changes affect Kv channel kinetics more closely in an isolated expression system. Initially, no significant chloride concentration-dependent effects on channel steady-state gating kinetics were observed. Only after disruption of the cytoskeleton with cytochalasin-D did we see significant chloride concentration-dependent shifts in gating kinetics. This suggests that the shift in gating kinetics is mediated through effects of intracellular chloride concentration on cytoplasmic domain tertiary conformation as cytoskeletal interaction appears to mask the effect. Furthermore, as cytoskeletal disruption only impacts channel gating kinetics at low physiological intracellular chloride concentrations, these studies highlight the importance of paying close attention to anion concentrations used under experimental conditions.

Introduction

Studies in rat hippocampal astrocytes [4] or salamander photoreceptors [25] demonstrate a Cl⁻-dependent reduction of outwardly rectifying voltage-gated potassium (Kv) currents. The reduction of Kv current in astrocytes was a result of Cl⁻ efflux following GABA_A receptor activation [3]. This phenomena is not merely restricted to potassium channels however. In salamander photoreceptors, activity-dependent [Cl⁻] changes have been shown to reduce voltage-gated Ca²⁺ channel activity which leads to reduced synaptic transmission at the first retinal synapse [24]. Further studies in photoreceptors also demonstrated effects of [Cl⁻] on Kv channels [25]. These Cl⁻-dependent effects were found to be consistent with the anions’ lyotropic or Hofmeister physical characteristics that are based on charge density and associated water of hydration relating to ease of anion approach to the protein–water interface. Thus, it was proposed that Cl⁻ may be screening charges on the protein structure leading to alterations in the field potential across the voltage sensor. An alternate explanation is that [Cl⁻]_i may indirectly affect gating by affecting the cytoplasmic domains of the channel.

Voltage-gated potassium channels are formed when four alpha subunits assemble to form a pore in a membrane dependent on an ∼120 amino acid cytosolic N-terminal domain, termed the tetramerization domain (T1). The T1 N-terminal domain hangs like a gondola directly below the channel [11], [30] and is highly conserved among the Kv1.x–Kv4.x α-subunits. What is interesting about this functional protein interaction domain is that in contrast to most assembly domain interfaces [14], this subunit interface is highly polar in nature, making the interaction much less stable. Mutagenesis studies addressing T1 complex stability and conformation showed that a conformational shift in the T1 tetramer is coupled to channel gating mechanisms [8], [18]. Because salts (anions in particular) play a large role in protein folding and conformation [5], [7], [13], it may be that [Cl⁻] is linked to Kv channel gating via the ability to affect the T1 tetramer conformational stability. To address this possibility, the effects of [Cl⁻]_i on individual Kv channel gating are assessed more closely using the isolated HEK293 cell expression system.