Conifer metabolite pisiferic acid restores activity in human Kv1.2 potassium channels carrying pathogenic sequence variants

Sequence variants in KCNA2, the gene encoding voltage-gated potassium channel Kv1.2, cause epilepsy, intellectual disability, and movement disorders. Drugs that directly correct mutant Kv1.2 function are lacking.  Kv1.2 downregulation is also implicated in pain and amyotrophic lateral sclerosis (ALS). We recently found that the abietane diterpenoid pisiferic acid (PA) from conifer Chamaecyparis pisifera beneficially restores activity in pathogenic loss-of-function (LOF)-variant Kv1.1 channels.  Here, using cellular electrophysiology, we classified 19 human Kv1.2 gene variants (pathogenic or of unknown significance) into LOF, gain of function (GOF), or mixed LOF/GOF. By hyperpolarizing their voltage dependence of activation, PA improved function in 13/13 LOF and 1/1 LOF/GOF pathogenic Kv1.2 variants tested, using cRNA ratios representative of autosomal dominant KCNA2 disorders. In silico docking, mutagenesis, and electrophysiology identified a PA binding site in the Kv1.2 volta..., Channel subunit cRNA preparation and Xenopus laevis oocyte injection
Wild-type Kv1.1 and wild-type and mutant Kv1.2 cDNAs were generated (Genscript, Piscataway, NJ) in the pMAX and pTLNx expression vectors.  As previously described, we generated cRNA transcripts encoding human Kv1.2 (wild-type and mutant) by in vitro transcription using the mMessage mMachine kit (Thermo Fisher Scientific), after vector linearization, from cDNA subcloned into plasmids (pMAX) incorporating Xenopus laevis β-globin 5’ and 3’ UTRs flanking the coding region to enhance translation and cRNA stability.  We injected defolliculated stage V and VI Xenopus laevis oocytes (Xenoocyte, Dexter, MI, US) with Kv1 cRNAs (0.1-10 ng). We incubated the oocytes at 16 oC in ND96 oocyte storage solution containing penicillin and streptomycin, with daily washing, for 1-2 days prior to two-electrode voltage-clamp (TEVC) recording.
Two-electrode voltage clamp (TEVC)
We performed TEVC at room temperature using an OC-725C..., ## Conifer metabolite pisiferic acid restores activity in human Kv1.2 potassium channels carrying pathogenic sequence variants

The datasets included are the original Excel files used to generate each panel for figures 2-9 in this manuscript. The title of each Excel file is labeled to directly correspond to the panel of each figure in the manuscript:

**Figure Number & panel > Channel Investigated (including mutant and wild type (WT)) > Condition > Parameter Measured.**

Example:

**Figure 2A, B - Kv1.2 12 uM Pisiferic acid - IV.xlsx**

The data contained within this repository are those obtained from cellular electrophysiology recordings. We used two-electrode voltage clamp (TEVC) electrophysiology and the *Xenopus laevis* oocyte expression system to record the electrical activity of wild-type Kv1.2 and Kv1.1/Kv1.2 channels in response to pisiferic acid. We also studied nineteen Kv1.2 disease-linked mutations. Using TEVC, we biophysically characterized homomeric homozygous patient Kv1....,