KCNH3 Loss-of-Function Variant Associated with Epilepsy and Neurodevelopmental Delay Enhances Kv12.2 Channel Inactivation

Figure A1. Window curves of functionally expressed homomeric WT, homomeric A371V, and heteromeric WT + A371V Kv12.2 channels. (A) Steady-state channel open probability curves (broken lines) were obtained by multiplying the respective activation and inactivation curves defined by the means of the Boltzmann parameter V1/2 and k determined in CHO cells in external solution containing 40 mM K+ (see Supplementary Table S1). (B) Overlay of window curves. WT Kv12.2 channels exhibit considerable steady-state open probability in the whole range of physiological membrane potentials with a maximal value of 0.56 at −25 mV. Co-expression of A371V yielded a maximum of 0.3 at −30 mV and homomeric A371V channels exhibit only marginal open probability with a maximum of 0.038 at −48 mV.

Figure 1. Functional characterization of WT and A371V Kv12.2 channels in Xenopus oocytes. (A) Two-electrode voltage clamp (TEVC) recordings from Xenopus oocytes performed in external solution containing 5 mM K+ four days after injection with a total of 5 ng Kv12.2 cRNA (WT only and A371V only), or 2.5 ng of WT and A371V each. Families of current traces were elicited with 1 s depolarizing voltage steps to potentials ranging between −80 and +60 mV to activate the Kv12.2 channels. Boxed left: Currents elicited at the onset of the depolarizing pulses on an expanded timescale to better illustrate the peak currents. Note that, in oocytes expressing the A371V Kv12.2 mutant alone, outward currents and inward tail currents are virtually absent. Boxed upper right: Overlayed current traces elicited with a potential step to +60 mV in oocytes expressing WT (black trace) and WT + A371V Kv12.2 channels (cyan). The current trace recorded from an oocyte co-expressing WT + A371V Kv12.2 was upscaled by a factor of 2.8 to the WT peak amplitude. Horizontal lines indicate zero current. Pulse protocol and recording conditions (TEVC, 5 K+) shown in the inset (lower right). (B) Xenopus oocyte currents recorded in…

Figure 2. Functional expression of WT and mutant Kv12.2 channels in CHO cells is enhanced by reduced culture temperature enabling biophysical analysis of the LoF mutant. (A) Distributions of current densities obtained with two culture conditions (37/37 °C or 37/32 °C) are given as boxplots for WT data (black) and WT + A371V co-expression data (cyan). Electrophysiological experiments were performed 48–60 h after transfecting CHO cells either with WT Kv12.2 channel cDNA alone or with a 1:1 ratio of WT + A371V cDNA, yielding the same total amount of channel cDNA. Part of the glass plates with transfected cells had been transferred to an incubator with a reduced temperature of 32 °C, 20 h after the transfection procedure. Membrane currents were recorded in 40 K+ external solution with an availability protocol as shown in (B). Tail current amplitudes at −100 mV were obtained from Boltzmann fits to tail current–prepulse voltage relationships. Tail current density was calculated by dividing the current amplitude by the cell capacitance. Number of experiments given in brackets. Black asterisks indicate a significant difference for reduced culture temperature compared to continuous maintenance at…

Figure 3. Voltage dependence and time course of WT and mutant Kv12.2 channel activation. (A) Membrane currents recorded in CHO cells in 40 K+ external solution using an activation protocol three days after transfection with WT or A371V Kv12.2 cDNA. Families of current traces were elicited by hyper- and depolarizing 4 s test pulses to potentials between −120 and +40 mV from a holding potential of −80 mV. The variable test pulses were followed by a final constant potential step to −110 mV to obtain the activation curves from tail current amplitudes. Note the differing current scales. Horizontal lines indicate zero current. (B) Normalized tail current amplitudes (means ± SEM) during the final constant pulse to –110 mV are plotted for WT (transfection and injection), A371V (transfection), and co-expression data (injection) against the preceding variable test pulse potential. Prior to averaging, data of individual experiments were normalized using the amplitude of Boltzmann fits to the data points. The lines show Boltzmann fits to the mean normalized data for WT (black), A371V (red), and WT + A371V co-expression (cyan). Number of experiments given in parentheses. (C,D) The time course of Kv12.2…

Figure 4. The A371V amino acid substitution drastically affects voltage dependence and time course of Kv12.2 channel inactivation. (A) Overlays of WT and A371V Kv12.2 current traces recorded in CHO cells in 40 K+ external solution with the indicated triple-pulse protocol. From a holding potential of −30 mV, a first pulse to +80 mV (P1) to activate and inactivate the channels was followed by a step to potentials between −140 and +100 mV of variable duration (P2) to induce recovery from inactivation with minimal deactivation. A final pulse to +80 mV (P3) reinitiated inactivation. P2 duration steadily increased with more positive potentials and differed for WT and mutant Kv12.2 channels to account for significant differences in the rate of recovery from inactivation (see Figure 2E). (B) Families of WT and A371V Kv12.2 current traces recorded with a slightly modified triple-pulse protocol. From a holding potential of −30 mV, a first pulse to +80 mV (P1) was followed by a constant step to −140 mV (P2) to induce recovery from inactivation in most channels. The variable P3 pulse to potentials between +80 and −20 mV served to assess the inactivation rate. Once more, P2 duration differed for WT and…