Mutation analysis of the hyperpolarization-activated cyclic nucleotide-gated channels HCN1 and HCN2 in idiopathic generalized epilepsy

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN1-4) channels play an important role in the regulation of neuronal rhythmicity. In the present study we describe the mutation analysis of HCN1 and HCN2 in 84 unrelated patients with idiopathic generalized epilepsy (IGE). Several functional variants were identified including the amino acid substitution R527Q in HCN2 exon 5. HCN2 channels containing the R527Q variant demonstrated a trend towards a decreased slope of the conductance-voltage relation. We also identified a variant in the splice donor site of HCN2 exon 5 that results in the formation of a cryptic splice donor. In HCN1, the amino acid substitution A881T was identified in one sporadic IGE patient but was not observed in 510 controls. Seven variants were examined further in a case-control association study consisting of a larger cohort of IGE patients. Further studies are warranted to more clearly establish the contribution of HCN1 and HCN2 dysfunction to the genetic variance of common IGE syndromes.

Figure 1

Location and evolutionary conservation of P42, A881, and R527. A. Schematic representation of HCN channels. The locations of the three amino acid substitutions identified in this study are shown. B. P42, located in the N-terminus of HCN1, is conserved in mammalian HCN1 channels. C. A881, located in the C-terminus of HCN1, is conserved in mammalian HCN1 channels. D. R527, located in the C-linker between the S6 segment and the CNBD, is invariant. Dots indicate identity to the top sequence. The alignments were generated using ClustalW1.82 (http://www.ebi.ac.uk/cgi-bin/clustalw).

Figure 2

Location and functional effect of the HCN2 intron 5 variant c.1584+7C>T. A. The substitution of the cytosine residue (c) at c.1584+7 with a thymine (t) residue generates a potential cryptic splice donor site that closely matches the sequence of the wild-type splice donor site. Use of the cryptic donor site will result in the extension of exon 5 (boxed) by five additional nucleotides. Transcripts containing the longer version of exon 5 will result in a premature stop codon (asterisk) in exon 6. B. Transcripts containing the longer version of exon 5 were detected by RT-PCR analysis and visualized in 2% agarose gels. No PCR amplification was observed from samples containing either the wild-type sequence (Lane 1) or the c.1584+7C>T variant (Lane 2) when reverse transcriptase was omitted from the cDNA synthesis. No PCR amplification was observed from samples containing only the wild-type splice donor site (Lanes 3 and 4). The expected 101-bp PCR product was observed from samples that contained the c.1584+7C>T variant (Lanes 5 and 6). Lane B represents the negative control containing no cDNA in the PCR reaction; Lane M represents the 50-bp molecular weight ladder (Invitrogen). C. Transcripts in which exon 5 was longer by 5 bp were confirmed by sequence analysis.

Figure 3

Functional analysis of homomeric mouse HCN2 channels, consisting of either wild-type or R500Q mutant subunits. (A) Currents from one representative patch of each channel construct are shown in the absence (black) and presence (red) of saturating (4 mM) cAMP. The currents were recorded in response to voltage pulses from a holding potential of 0 mV to test potentials between −70 mV and −150 mV, returning to a tail potential of −40 mV. (B) Conductance-voltage relations were obtained from normalized tail currents, either for HCN2 wild-type (filled symbols) or mutant (open symbols). The data shown are mean normalized values ± SEM and are fit with a Boltzmann relation.

Figure 4

HCN2 C-terminal region crystal structure. Structure of the HCN2 C-terminal region (Zagotta et al., 2003) viewed from the side (left) and from the membrane (right). The structure is positioned below the membrane-spanning portion of the channel, as it is thought to be in vivo. The structure contains four subunits, two in dark gray and two in light gray, with the C-linkers making up the top half of the structure and the CNBDs the bottom half. cAMP (yellow) is bound in the CNBD of each subunit. Residues at position 500 (orthologous to position 527 in human sequence) and the salt bridge residues on the B′ and D′ helices, as well as the β-roll of the CNBDs, are shown in CPK format: R500 (light blue), K472 (blue), E502 and D542 (both in red). Side view of one subunit (inset). The A′-D′ helices of the C-linker are labeled, and the R500 residue can be seen projecting towards the hole down the center of the structure.