An N-terminal deletion variant of HCN1 in the epileptic WAG/Rij strain modulates HCN current densities.

Konstantin Wemhöner,Michael F Netter,Aytug K Kiper,Tatyana Kanyshkova,Thomas Budde,Nicole Silbernagel,Sven G Meuth,Stefan Bittner,Niels Decher,Juncal Fernandez-Orth,Susanne Rinné

doi:10.3389/fnmol.2015.00063

Abstract

Rats of the Wistar Albino Glaxo/Rij (WAG/Rij) strain show symptoms resembling human absence epilepsy. Thalamocortical neurons of WAG/Rij rats are characterized by an increased HCN1 expression, a negative shift in Ih activation curve, and an altered responsiveness of Ih to cAMP. We cloned HCN1 channels from rat thalamic cDNA libraries of the WAG/Rij strain and found an N-terminal deletion of 37 amino acids. In addition, WAG-HCN1 has a stretch of six amino acids, directly following the deletion, where the wild-type sequence (GNSVCF) is changed to a polyserine motif. These alterations were found solely in thalamus mRNA but not in genomic DNA. The truncated WAG-HCN1 was detected late postnatal in WAG/Rij rats and was not passed on to rats obtained from pairing WAG/Rij and non-epileptic August Copenhagen Irish rats. Heterologous expression in Xenopus oocytes revealed 2.2-fold increased current amplitude of WAG-HCN1 compared to rat HCN1. While WAG-HCN1 channels did not have altered current kinetics or changed regulation by protein kinases, fluorescence imaging revealed a faster and more pronounced surface expression of WAG-HCN1. Using co-expression experiments, we found that WAG-HCN1 channels suppress heteromeric HCN2 and HCN4 currents. Moreover, heteromeric channels of WAG-HCN1 with HCN2 have a reduced cAMP sensitivity. Functional studies revealed that the gain-of-function of WAG-HCN1 is not caused by the N-terminal deletion alone, thus requiring a change of the N-terminal GNSVCF motif. Our findings may help to explain previous observations in neurons of the WAG/Rij strain and indicate that WAG-HCN1 may contribute to the genesis of absence seizures in WAG/Rij rats.

Highlights

Alterations in hyperpolarization-activated cyclic nucleotidegated cation channel (HCN) channel expression and the corresponding pacemaker current hyperpolarization-activated inward current (Ih) were attributed to pathological activity in the thalamocortical (TC) system, like spike-and-wave discharge (SWD) which are typical for childhood absence epilepsy (Budde et al, 2005; Kuisle et al, 2006; Biel et al, 2009; Kanyshkova et al, 2012)
While cloning of HCN1 channels from cDNA libraries obtained from thalamus of adult rats, we observed that the HCN1 channel from Wistar Albino Glaxo/Rij (WAG/Rij) rats harbors an N-terminal deletion (Figure 1)
Sequencing of genomic DNA from the thalamus of WAG/Rij rats revealed that this deletion was not present on chromosome 2 when compared to the genomic sequence of Rattus norvegicus (NW_047620; Figure 1B)

Summary

Introduction

Alterations in HCN channel expression and the corresponding pacemaker current Ih were attributed to pathological activity in the thalamocortical (TC) system, like SWD which are typical for childhood absence epilepsy (Budde et al, 2005; Kuisle et al, 2006; Biel et al, 2009; Kanyshkova et al, 2012). In WAG/Rij rats, systemic injections of Ih blockers resulted in a dose-dependent decrease in SWD pointing to a crucial role of HCN channels (van Luijtelaar et al, 2011b; van Luijtelaar and Zobeiri, 2014). The expression of HCN1 was higher in WAG/Rij rats than in non-epileptic rat strains (Budde et al, 2005; Kanyshkova et al, 2012). This change was accompanied by an increased Ih current density, a negative shift of the activation curve of Ih, and a decreased sensitivity of Ih to cAMP in TC neurons which might result in an impairment to control the shift from burst to tonic firing. We have functionally characterized WAG-HCN1 by heterologous expression and revealed a possible contribution of this HCN1

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