Amyloid β peptide as a physiological modulator of neuronal ‘A’-type K+ current

doi:10.1016/j.neurobiolaging.2005.09.038

Neurobiology of Aging

Volume 27, Issue 11, November 2006, Pages 1673-1683

https://doi.org/10.1016/j.neurobiolaging.2005.09.038 Get rights and content

Abstract

Control of neuronal spiking patterns resides, in part, in the type and degree of expression of voltage-gated K⁺ channel subunits. Previous studies have revealed that soluble forms of the Alzheimer's disease associated amyloid β protein (Aβ) can increase the ‘A’-type current in neurones. In this study, we define the molecular basis for this increase and show that endogenous production of Aβ is important in the modulation of Kv4.2 and Kv4.3 subunit expression in central neurones.

A-type K⁺ currents, and Kv4.2 and Kv4.3 subunit expression, were transiently increased in cerebellar granule neurones by the 1–40 and 1–42 forms of Aβ (100 nM, 2–24 h). Currents through recombinant Kv4.2 channels expressed in HEK293 cells were increased in a similar fashion to those through the native channels. Increases in ‘A’-type current could be prevented by the use of cycloheximide and brefeldin A, indicating that protein expression and trafficking processes were altered by Aβ, rather than protein degredation.

Endogenous Aβ production in cerebellar granule neurones was blocked using inhibitors of either γ- or β-secretase and resulted in decreased K⁺ current. Crucially this could be prevented by co-application of exogenous Aβ (1 nM), however, no change in Kv4.2 or Kv4.3 subunit expression occurred.

These data show that Aβ is a modulator of Kv4 subunit expression in neurones at both the functional and the molecular level. Thus Aβ is not only involved in Alzheimer pathology, but is also an important physiological regulator of ion channel expression and hence neuronal excitability.

Introduction

Amyloid β protein (Aβ) is the major constituent of senile plaques found in the brain of Alzheimer's disease patients [8] and is widely regarded as a neurotoxin, key to the neurodegenerative process in Alzheimer's disease (AD; reviewed by [1]). However, Aβ is constitutively derived, throughout life, via the highly regulated and sequential β- and γ-secretase cleavage of amyloid precursor protein (APP) and can be measured at nanomolar levels in the cerebrospinal fluid of both AD sufferers and their age-matched controls [20]. Furthermore, Aβ has been detected in culture medium in which neuronal cultures have been grown [12]. This suggests the possibility that Aβ has a role to play in normal brain function.

Several previous studies suggest that Aβ can alter the expression of ion channels in central neurones [4], [15], [23], [24], [25], [33]. In cerebellar granule neurones we have found that the rapidly inactivating or ‘A’ type current (I_KA) is increased by the application of Aβ at concentrations of 10–1000 nM for 24 h [25]. This current has been suggested to be mediated by members of the Shal related family of voltage-gated K⁺ channel α-subunits known as Kv4.2 and Kv4.3. Currents encoded by these subunits are activated at sub-threshold potentials and define excitability by preventing the back propagation of action potentials and controlling the frequency of slow repetitive spike firing [26]. The alteration of K⁺ channel expression by incubation with low levels of exogenous, synthetic Aβ suggests the possibility that endogenous Aβ may act as a physiological regulator of the expression of these functionally important channels.

The recent development of inhibitors of γ- and β-secretase now provide us with the opportunity to assess the importance of endogenous production of amyloid β protein in expression of I_KA. In this study, we have extended our examination of the effects of exogenous Aβ on expression of I_KA in central neurones and have probed the effects of blocking endogenous Aβ production on both the functional expression of I_KA, and on the levels of Kv4 channel subunits. This report provides, for the first time, both functional effects and identification of the specific Kv subunits upregulated by Aβ.

Section snippets

Cell culture

All experiments were performed using either primary cultures of rat cerebellar granule neurones or HEK 293 cells stably transformed to express Kv4.2 subunits. For primary cultures, cells were obtained by enzymatic and mechanical dissociation as previously described [13], [25]. Briefly, tissue was removed from 6 to 8-day-old rat pups and triturated following a 15 min trypsin (2.5 mg ml⁻¹ in phosphate buffered saline) digestion. Trypsin digestion was halted by the addition of a solution containing

Aβ peptides enhance K⁺ currents by increasing the number of Kv4 α-subunits

Exogenous Aβ_1–40, applied to cultured rat cerebellar granule neurones for 24 h, induces a concentration-dependent increase in the ‘A’-type K⁺ channel current [25]. This increase occurs in the range of concentrations greater than 1 nM Aβ. Our subsequent studies have defined the time course of this effect using a maximally effective concentration of Aβ_1–40 (100 nM). Peak current–voltage relationships for cells preincubated with either Aβ_1–40 or the reverse sequence of the peptide (Aβ_40–1) as a

Discussion

In our previous study, we found that a 24 h preincubation of cells with soluble (but not aggregated) Aβ_1-40 at concentrations between 10 nM and 1 μM resulted in a concentration-dependent increase in I_KA with no effect on I_KV [25]. Whilst those data appeared to contradict observations of Yu et al. and Colom et al. [33], [4] reported a selective increase in a non-inactivating component of K⁺ current following application of synthetic Aβ, they used much higher, non-physiological concentrations of

Acknowledgements

This work was funded by the MRC, Wellcome Trust, Alzheimer's Society and Alzheimer's Research Trust. We thank Drs. Lyanne Schlichter and Wei Wong, Toronto Western Research Institute, Canada for their generous gift of ECFP tagged Kv4.2.

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¹: Present address: Department of Pediatrics and Institute for Molecular Pediatric Sciences, University of Chicago, Pritzker School of Medicine, 5841 S. Maryland Avenue, Chicago, IL 60637, USA.

²: Present address: Department of Neurology, University of Minnesota, 420 Delaware Street SE, MMC #295, Minneapolis MN 55455, USA.

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Amyloid β peptide as a physiological modulator of neuronal ‘A’-type K+ current

Abstract

Introduction

Section snippets

Cell culture

Aβ peptides enhance K+ currents by increasing the number of Kv4 α-subunits

Discussion

Acknowledgements

Brain Res

Life Sci

Biochem Biophys Res Commun

Biophys J

J Biol Chem

FEBS Lett

Brain Res

Neuron

J Biol Chem

Brain Res

Neuroscience

Neurobiol Dis

Neuron

Neurobiol Dis

A cell biological perspective on Alzheimer's disease

Annu Rev Cell Dev Biol

Amyloid beta protein is internalized selectively by hippocampal field CA1 and causes neurons to accumulate amyloidogenic carboxyterminal fragments of the amyloid precursor protein

J Comp Neurol

Amyloid β peptide as a physiological modulator of neuronal ‘A’-type K⁺ current

Aβ peptides enhance K⁺ currents by increasing the number of Kv4 α-subunits