A Germline Variant in the PANX1 Gene Has Reduced Channel Function and Is Associated with Multisystem Dysfunction

Abstract

Pannexin1 (PANX1) is probably best understood as an ATP release channel involved in paracrine signaling. Given its ubiquitous expression, PANX1 pathogenic variants would be expected to lead to disorders involving multiple organ systems. Using whole exome sequencing, we discovered the first patient with a homozygous PANX1 variant (c.650G→A) resulting in an arginine to histidine substitution at position 217 (p.Arg217His). The 17-year-old female has intellectual disability, sensorineural hearing loss requiring bilateral cochlear implants, skeletal defects, including kyphoscoliosis, and primary ovarian failure. Her consanguineous parents are each heterozygous for this variant but are not affected by the multiorgan syndromes noted in the proband. Expression of the p.Arg217His mutant in HeLa, N2A, HEK293T, and Ad293 cells revealed normal PANX1 glycosylation and cell surface trafficking. Dye uptake, ATP release, and electrophysiological measurements revealed p.Arg217His to be a loss-of-function variant. Co-expression of the mutant with wild-type PANX1 suggested the mutant was not dominant-negative to PANX1 channel function. Collectively, we demonstrate a PANX1 missense change associated with human disease in the first report of a "PANX1-related disorder."

Keywords: ATP; cell biology; cell surface protein; development; pannexin.

FIGURE 1.

Three generation pedigree demonstrating consanguinity, with the proband's parents as first cousins. Selected clinical conditions are noted. Full shading represents the homozygous R217H variants, and half-shading represents heterozygous R217H PANX1 variants. Arrow indicates proband; “+” indicates wild-type allele.

FIGURE 2.

Disease-linked R217H mutant exhibits no characteristic difference in cellular localization or glycosylated isoforms in comparison with wild-type PANX1. A, N2A and NRK cells were engineered to express wild-type PANX1 or the R217H mutant and immunostained for the location of PANX1 (red) or the gap junction protein, Cx43 (green). Arrow indicates the R217H mutant at the cell surface with no apposing cells. Nuclei were stained with TO-PRO®-3 (blue). Bars, 10 μm. B, untransfected N2A, 293T, and Ad293 cells or cells expressing wild-type PANX1 or the R217H mutant were immunoblotted for PANX1 or the gel loading control β-tubulin. Note that all glycosylated species of wild-type PANX1 and the R217H mutant (Gly0, Gly1, and Gly2) are expressed. Molecular mass markers are shown, and the experiments were repeated across a minimum of three cell lines to eliminate any cell type differences that might exist. C, model of PANX1 illustrating the approximate topological position of the R217H variant (red sphere).

FIGURE 3.

R217H mutant exhibits defective dye uptake and ATP release. A, untransfected Ad293 cells or cells expressing wild-type PANX1 or the R217H mutant were immunolabeled for PANX1 (red), and all cells were counterstained for TO-PRO®-3 (blue). Bar, 10 μm. B, untransfected (UNTR) Ad293 cells or cells expressing wild-type PANX1 or R217H, or expressing wild-type PANX1 and treated with probenecid were subjected to ethidium bromide (EtBr) dye uptake over a period of 40 min. Mean fluorescent measurements revealed that wild-type PANX1-expressing cells were capable of dye uptake, whereas R217H-expressing, untreated cells, or cells expressing wild-type PANX1 and treated with probenecid exhibited significantly reduced dye uptake. *, p < 0.05; ***, p < 0.001, n = 3. C, untransfected (UNTR) N2A cells or N2A cells expressing wild-type PANX1 or the R217H mutant were assayed for ATP release upon treatment with a high potassium (K⁺) medium containing or lacking the channel blocker CBX. ns, not significant. **, p < 0.01, n = 3.

FIGURE 4.

R217H mutant shows a dramatic reduction in channel function when expressed in HEK293T cells. A, representative current-voltage relationships recorded in the absence or presence of CBX in cells expressing wild-type PANX1 (top) and the R217H mutant (bottom). B, summary of ramp currents recorded at +100 mV from wild-type PANX1 and R217H mutant expressing or mock-transfected (UNTR) HEK293T cells. C, ramp currents at −60 mV recorded in HEK293T cells expressing wild-type PANX1 and the R217H mutant, before (control) and after treatment with high potassium (High K⁺) or high potassium and CBX. D, representative Western blot reveals comparable cellular expression of wild-type PANX1 and R217H channels in whole-cell lysates prepared from sister HEK293T cells used in electrophysiological recordings (position of molecular mass standards are shown). For all panels, the number of cells recorded is indicated in parentheses. p values were calculated in comparison with PANX1 groups using two-way ANOVA analyses with Bonferroni post-tests (B and C), ***, p < 0.001.

FIGURE 5.

R217H mutant is not dominant-negative to co-expressed PANX1. A, representative current-voltage relationships recorded in HEK293T cells expressing PANX1, R217H, or both. B, summary of ramp currents recorded at +100 mV from wild-type PANX1, R217H, or PANX1/R217H co-expressing cells. C, ramp currents at −60 mV recorded in HEK293T cells expressing wild-type PANX1, the R217H mutant, or both, before (control) and after treatment with high potassium (High K⁺) or high potassium and CBX. For all panels, the number of cells recorded is indicated in parentheses. *, p < 0.05; **, p < 0.01. ns, not significant.

FIGURE 6.

A, cell surface biotinylation of wild-type PANX1 and the R217H mutant in the presence and absence of high potassium. Cells were not transfected (NT) or transiently transfected with cDNA encoding either wild-type PANX1 or the R217H mutant. Forty eight hours after transfection, cells were untreated (−) or treated (+) with 140 mm K⁺Glu in DMEM for 40 min. Cells were placed on ice, washed in ice-cold Hanks' balanced salt solution, and cell surface proteins biotinylated. Biotinylated proteins were precipitated using neutravidin-conjugated beads, subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted (IB) for PANX1 or GAPDH. Note the abundance of cell surface biotinylated PANX1 and the R217H mutant, although the lack of biotinylated GAPDH indicates that only cell surface proteins were biotinylated. Position of molecular mass standards are shown. B, R217H mutant showed a dramatic reduction in channel function when expressed in HEK293T cells pretreated with BFA for 6 h while CBX effectively blocked channel function. C, after pre-treatment with BFA for 6 h, ramp currents at −60 mV were recorded in HEK293T cells expressing wild-type PANX1 or the R217H mutant, before (control) and after treatment with high potassium (High K⁺) or high potassium and CBX. n = 6 for each panel. ***, p < 0.001.