Authors :
Presenting Author: Alfred George, MD – Northwestern University Feinberg School of Medicine
Chris Thompson, PhD – Northwestern University; Reshma Desai, M.S. – Northwestern University; esther Cha, B.S. – Northwestern University; Alexandra Hong, B.S. – Northwestern University; Carlos Vanoye, PhD – Northwestern University
Rationale:
KCNH1
variants are associated with neurodevelopmental disorders including Zimmermann-Laband and Temple-Baraitser syndromes that feature epilepsy, autism, intellectual disability and developmental delay. KCNH1 encodes the voltage-gated potassium channel KV10.1 (also known as EAG1), which is expressed in glutamatergic neurons and activates at subthreshold potentials to help stabilize the resting membrane potential (RMP). Currently, there more than 50 published KCNH1 variants, but few have been investigated for their functional consequences. Methods:
We used HEK293T cells to heterologously express wild-type (WT) or variant K
V10.1 and investigated functional properties of 6 population variants and 24 disease-associated variants. Whole-cell potassium currents were recorded by automated voltage clamp. Additionally, we used whole-cell current clamp to measure RMP in cells expressing WT or variant K
V10.1.
Results:
We investigated the functional properties of 30
KCNH1 variants including 24 disease-associated variants and six population variants. Among the 24 disease-associated variants, we observed that whole-cell potassium current density was largely unaffected. However, five variants (K354E, K354N, V383L, G496E, and G496R) showed significantly smaller steady-state currents, while one variant (G375R) showed significantly larger steady-state currents. However, 14 of 24 disease-associated variants showed a larger ratio of instantaneous to steady-state current, suggesting that these channels open instantaneously. Additionally, 14 of the 24 exhibited large hyperpolarized shifts in the voltage-dependence of activation. Taken together, this combination of biophysical defects suggest that the majority of disease-associated variants have predominantly gain-of-function effects. Importantly, all of the population variants had functional properties indistinguishable from WT channels. We also investigated the effects of some disease-associated variants on RMP. Expression of WT-
KCNH1 hyperpolarized the RMP of HEK cells by approximately 30 mV. Expression of variants R357Q or R357P induced approximately 50 mV hyperpolarization of the RMP. However, the RMP of cells expressing the population variant S819T was indistinguishable from WT.
Conclusions:
We demonstrated that automated patch-clamp recording can be successfully applied to the functional evaluation of
KCNH1 variants. While gain-of-function may be a common feature determined from voltage-clamp experiments, hyperpolarization of the RMP may be the shared physiological consequence, which we predict translates to dampened neuronal excitability.
Funding:
This work was funded by NIH grant NS108874