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Brain Commun
2023 Jan 01;53:fcad156. doi: 10.1093/braincomms/fcad156.
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Cation leak: a common functional defect causing HCN1 developmental and epileptic encephalopathy.
McKenzie CE
,
Forster IC
,
Soh MS
,
Phillips AM
,
Bleakley LE
,
Russ-Hall SJ
,
Myers KA
,
Scheffer IE
,
Reid CA
.
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Pathogenic variants in HCN1 are an established cause of developmental and epileptic encephalopathy (DEE). To date, the stratification of patients with HCN1-DEE based on the biophysical consequence on channel function of a given variant has not been possible. Here, we analysed data from eleven patients carrying seven different de novo HCN1 pathogenic variants located in the transmembrane domains of the protein. All patients were diagnosed with severe disease including epilepsy and intellectual disability. The functional properties of the seven HCN1 pathogenic variants were assessed using two-electrode voltage-clamp recordings in Xenopus oocytes. All seven variants showed a significantly larger instantaneous current consistent with cation leak. The impact of each variant on other biophysical properties was variable, including changes in the half activation voltage and activation and deactivation kinetics. These data suggest that cation leak is an important pathogenic mechanism in HCN1-DEE. Furthermore, published mouse model and clinical case reports suggest that seizures are exacerbated by sodium channel blockers in patients with HCN1 variants that cause cation leak. Stratification of patients based on their 'cation leak' biophysical phenotype may therefore provide key information to guide clinical management of individuals with HCN1-DEE.
Figure 1.
Functional analysis of previously characterised HCN1 pore domain variants revealed significant cation leak (A) Location of variants analysed in this study (red spheres) within the structure of HCN1. Variants with previously published functional analysis shown in red text, and variants without prior functional analysis shown in blue text. The S4 helix of the voltage sensing domain (VSD) is shown is dark blue, and the S5 and S6 helices of the pore domain (PD) are shown in orange and light blue, respectively. The structure is based on the depolarised (closed) conformation of HCN126 and was rendered using PyMOL (The PyMOL Molecular Graphics System, Version 2.3.4 Schrödinger, LLC). (B) Representative voltage clamp data from oocytes expressing HCN1 wild-type (WT) and co-expressed WT + M305L (top); in the presence of CsCl (middle) and with the CsCl traces subtracted from the corresponding traces at each test potential (bottom). Each dataset shows current traces in response to a series of 10 mV voltage steps (inset) from the holding potential (-30 mV) to test potentials in the range -120 mV to +20 mV. (C) An expanded view of HCN1 WT and co-expressed WT + S272P, WT + M305L, WT + I380F, WT + G391D, and WT + S399P traces with capacitive charging transients eliminated by subtracting records in Cs+ solution (see Materials and methods) and thus reveal the instantaneous and time-dependent activating components associated with the heterologously expressed channels. Arrows indicate instantaneous current component (Iinst) for -120 mV step. Dotted line indicates 0 current reference. (D) Expanded view of WT + I380N and WT + A387S traces, with the same properties as those in (C). (E) Instantaneous current (normalised to steady-state current) at -100 mV for HCN1 WT and co-expressed WT + S272P, WT + M305L, WT + I380N, WT + I380F, WT + A387S, WT + G391D, and WT + S399P. P < 0.05 were considered significant and denoted *. Data were compared using one-way ANOVA with Dunnett’s post-hoc, compared to WT (See Supplementary Table 1 for detailed analysis and exact P-values).
Figure 2.
Functional characterisation of two novel HCN1 pore domain variants also revealed cation leak (A) Pooled current–voltage (I–V) data normalised to -120 mV shows markedly weaker inward rectification for co-expressing oocytes [WT + S272P (n = 9), WT + M305L (n = 10), WT + I380N (n = 10), WT + I380F (n = 7), WT + A387S (n = 9), WT + G391D (n = 5), and WT + S399P (n = 7)] compared with WT (n = 10). Grey rectangle highlights a range of voltages at which a typical neuron. (B) Average of raw steady-state current (Iss) for WT + variant HCN1 channels measured at end of test pulse to -120 mV. (C) Normalised instantaneous tail currents for the co-injected variants indicated in (B). Data points were fit with a single Boltzmann function (see Materials and methods). (D) Half activation voltages (V0.5) for HCN1 WT and WT+ variant, respectively, reported by Boltzmann fits to data in (C). (E) Probability of channels being open at -50 mV for HCN1 WT compared to co-expressed WT+ variant obtained from normalised Boltzmann fits to data in (C). (F) Representation of the effective charge (z) reported by the Boltzmann fit for HCN1 WT and WT+ variant. (G) Mean activation time constant obtained by fitting the time-dependent component of activating current from wild-type and WT+ variant oocytes with a single-exponential function (see Materials and methods). (H) Deactivation time constant at potentials between -90 and +50 mV obtained by fitting the time-dependent component of deactivating current from wild-type and WT+ variant oocytes with a single-exponential function (see Materials and Methods). P < 0.05 were considered significant and denoted *. Data were compared using one-way ANOVA with Dunnett’s post-hoc, compared to WT (See Supplementary Tables 1–4 for detailed analyses and exact P-values).
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