Functional characterisation of KCNA5 gene variants linked to pulmonary arterial hypertension

Extract

Pulmonary arterial hypertension (PAH) is a progressive pulmonary vascular disease with heritable forms that account for approximately 10–20% of cases [1]. Beyond variants in the transforming growth factor-β signalling pathway (BMPR2), mutations in ion channel genes have emerged as impactful contributors to disease pathogenesis [2]. Potassium channels are key regulators of pulmonary arterial smooth muscle cell (PASMC) membrane potential and calcium homeostasis, and their dysfunction promotes vasoconstriction and proliferation [3].

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Functional analysis of a rare KCNA5 variant (p.Gly435Arg) supports its reclassification as likely pathogenic in pulmonary arterial hypertension https://bit.ly/4cUu3oB

To the Editor:

Pulmonary arterial hypertension (PAH) is a progressive pulmonary vascular disease with heritable forms that account for approximately 10–20% of cases [1]. Beyond variants in the transforming growth factor-β signalling pathway (BMPR2), mutations in ion channel genes have emerged as impactful contributors to disease pathogenesis [2]. Potassium channels are key regulators of pulmonary arterial smooth muscle cell (PASMC) membrane potential and calcium homeostasis, and their dysfunction promotes vasoconstriction and proliferation [3].

In PAH, the voltage-gated potassium channel Kv1.5, encoded by KCNA5, has been shown to exhibit reduced expression and/or function [4, 5]. This has been confirmed in experimental pulmonary hypertension models. In PAH cohorts, several rare KCNA5 variants have been reported; however, due to the lack of robust functional validation, their pathogenic relevance is unclear, and most have been classified as variants of uncertain significance [6].

To fill this gap, we systematically evaluated KCNA5 variants by combining genetic screening in a large international PAH cohort with electrophysiological, structural and cellular analysis of selected variants. Patients with precapillary pulmonary hypertension were recruited from eight PAH expert centres in Germany and Austria, where clinical, haemodynamic, and follow-up data were collected. The study was approved by the ethics committee of the Medical Faculty of Heidelberg University (S-426/2017), and all participants provided written informed consent.

Targeted next-generation sequencing of established PAH-associated genes, including KCNA5, was performed in all patients as previously described [7]. We identified 11 rare KCNA5 variants, eight of which had not been previously reported. Six novel non-synonymous variants (five missense mutations and one in-frame deletion) were prioritised for functional investigation, while synonymous (not altering the protein sequence) and previously fully characterised variants [5, 8, 9] were not analysed (figure 1a). The cohort included 540 PAH patients with a median age of 57 years (range 17–77 years) at diagnosis, a female:male ratio of 4.3:1, and a mean pulmonary arterial pressure of 39 mmHg (range 25–90 mmHg). The clinical characteristics of the 21 variant carriers were comparable to the other patients.

FIGURE 1.

a) Workflow for identification and analysis of KCNA5 variants. Created with BioRender.com. b) Electrophysiological characterisation of Kv1.5 variants expressed in Xenopus laevis oocytes. Peak current amplitudes measured at +60 mV are shown for wildtype (WT) and each channel variant (N=3; n=16–19 oocytes per group). Data were analysed using the Kruskal–Wallis test followed by Dunn's multiple comparisons test. The G435R variant exhibited markedly reduced current amplitude compared to WT (p=0.0002). c) Single-channel recording of WT and G435R Kv1.5 channels. Representative recordings from excised inside-out patches of Xenopus oocytes expressing WT or G435R Kv1.5 channels are shown. Current levels at +40 mV corresponding to 0–3 simultaneously open channels are indicated. Mean open times were calculated from multiple patches (n=5–6 per group). G435R channels exhibited significantly shorter mean open times (τ_o) compared with WT (unpaired t-test, p=0.0016). d) Haploinsufficiency of G435R assessed using concatenated Kv1.5 dimer constructs. Peak current amplitudes at +60 mV are shown for homodimeric (WT–WT, G435R–G435R) and heterodimeric (WT–G435R) channels (N=2; n=23–24 per group). The schematic illustrates tetramer assembly from tandem dimer constructs (created with BioRender.com). Data were analysed using the Kruskal–Wallis test followed by Dunn's post hoc test (WT–WT versus G435R–G435R, p<0.0001; WT–WT versus WT–G435R, p=0.0139; WT–G435R versus G435R–G435R, p<0.0001). e) Structural context of residue Gly435 within Kv1.5. The predicted structure of the human Kv1.5 channel was obtained from the AlphaFold model and visualised with PyMOL software. The four α-subunits are shown in distinct colours. Grey planes indicate the approximate lipid bilayer boundaries. The mutated residue (G435R) is highlighted in red (space-filling representation) and shown in a magnified inset. f) Effect of Kv1.5 expression on proliferation rate of human pulmonary arterial smooth muscle cells (PASMCs). PASMC with no Kv1.5 expression (virus control), compared to PASMC overexpressing WT Kv1.5 and PASMC overexpressing the G435R variant. Cells were transduced with lentiviral vectors encoding WT-Kv1.5, G435R-Kv1.5 or virus control. Proliferation was assessed in 96-well plates, each point represents one well. Data were pooled from independent experiments. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn's multiple comparisons test (control versus WT, p<0.0001; control versus G435R, p=0.1429; WT versus G435R, p=0.0388). g) Localisation within the KCNA5 gene of Kv1.5 variants associated with pulmonary arterial hypertension. Variants on the upper side of the gene schematic were identified in our cohort; variants on the lower side represent previously reported KCNA5 variants. Only the variants highlighted in red have been shown to significantly reduce channel function. PAH: pulmonary arterial hypertension; ns: nonsignificant; *: p<0.05. Created with BioRender.com.

To assess the functional effects of the selected six rare Kv1.5 variants (ΔD72_P82, R87Q, D166A, P286L, G435R and T611K), each variant was cloned, expressed in Xenopus laevis oocytes, and K⁺ currents were measured using two-electrode voltage-clamp [10]. Frogs were handled in accordance with the Institutional Animal Care Committee guidelines of Semmelweis University. All variants, except G435R, produced K⁺ currents comparable to the wildtype (WT) (figure 1b). In contrast, oocytes expressing G435R showed a marked reduction in current amplitude, with an 87±1.3% decrease at +60 mV (p=0.0002) (figure 1b). Due to its pronounced loss-of-function, G435R was selected for further biophysical and functional characterisation.

The heterozygous G435R carrier was a 73-year-old female with idiopathic PAH presenting with decompensated right heart failure. She presented with no acute response to inhaled nitric oxide but a favourable clinical response to oral PAH combination therapy, later de-escalated to tadalafil monotherapy due to side-effects. Her comorbidities were intermittent atrial fibrillation, diabetes mellitus, mild renal insufficiency, and mild liver cirrhosis, and a severe hip arthrosis, which limited her 6-min walk distance.

Western blot analysis of membrane-enriched fractions revealed comparable Kv1.5 protein levels between WT and mutant channels (data not shown), ruling out differences in expression as the cause for the pronounced reduction in macroscopic Kv1.5 currents observed for G435R. To uncover the mechanistic basis of this loss-of-function, we performed single-channel recordings, which allow direct assessment of intrinsic gating behaviour [11]. The mean open time of G435R was significantly shorter than WT (12.9±0.7 ms versus 27.2±3.8 ms; p<0.01), explaining the severely reduced whole-cell currents and demonstrating that impaired gating, rather than permeation, underlies the functional impairment (figure 1c).

Because Kv1.5 channels assemble as tetramers, we modelled the heterozygous condition using tandem concatemers to enforce defined subunit stoichiometry (figure 1d). As expected, mutant–mutant concatemers showed a marked reduction in current amplitude at +60 mV compared to WT–WT (93.3±0.6%). Importantly, heterodimeric WT–G435R concatemers exhibited a significant reduction in current amplitude (53±3% of WT–WT; p=0.0139), indicating haploinsufficiency under heterozygous conditions.

To explore the structural basis of the G435R loss-of-function, we analysed the human Kv1.5 channel using the AlphaFold model. G435 is located near the intracellular end of the S5 helix, adjacent to the S4–S5 linker, a region critical for electromechanical coupling and channel gating (figure 1e). Substitution of glycine with the bulky, positively charged arginine is predicted to disrupt local interactions and impair electromechanical coupling, consistent with our observed gating defect. The marked shortening of mean open time suggests destabilisation of the open state rather than impaired channel expression or trafficking. Given the central role of Kv1.5 in regulating PASMC membrane potential and proliferation, we next examined the cellular consequences of the G435R variant.

To evaluate the impact of G435R at the cellular level, human PASMCs were isolated from donor lungs not used for transplantation (approved by the ethics committee of the Medical University of Vienna; IRB 976/2010), cultured under standard conditions as previously described [12], and infected with lentivirus encoding either WT Kv1.5 (WT), G435R Kv1.5 (G435R), or an empty vector (control), according to the manufacturer's instructions.

Overexpression of Kv1.5 (WT) significantly reduced PASMC proliferation (p<0.0001) (figure 1f), consistent with its known anti-proliferative effect. In contrast, overexpression of G435R failed to suppress proliferation and showed increased proliferation as compared to WT (p=0.14 versus control; and p=0.039 versus WT). This suggests that G435R interferes with the anti-proliferative function of Kv1.5, confirming a direct cellular link with PAH pathophysiology.

Some limitations of the study should be acknowledged. The small number of KCNA5 variant carriers limits genotype–phenotype correlations, and no first-degree relatives were available for segregation analysis. Functional assays were performed under basal conditions. The concatemer model enforces a fixed WT:mutant ratio and may not fully reflect in vivo assembly. Larger multicentre studies are needed to further clarify the role of KCNA5 in PAH and guide potential Kv1.5-targeted therapies.

In conclusion, we identified 11 rare KCNA5 variants in this multicentre, well-phenotyped pulmonary hypertension cohort, and assessed six for their functional impact. G435R emerged as likely pathogenic, being absent from large population databases (>60 000 controls) and consistently demonstrating loss-of-function across multiple complementary electrophysiological and cellular assays. Together, these findings fulfil American College of Medical Genetics and Genomics criteria PS3 (strong functional evidence), PM1 (critical domain), and PM2 (absence from controls), supporting reclassification of G435R as a likely pathogenic variant. Although Kv1.5 contributes to atrial repolarisation, the G435R carrier presented primarily with decompensated idiopathic PAH, and her intermittent atrial fibrillation may represent a secondary comorbidity rather than a primary manifestation of channel dysfunction [13].

As shown in figure 1g, our study expands the published spectrum by eight variants to a total of 34. G435R represents one of only three rare KCNA5 variants showing a significant functional impact. This is consistent with its localisation within a functionally critical region of the channel. These findings highlight the importance of systematic multi-platform validation for interpreting rare variants in vascular channelopathies.