How do Kir3.4 mutations cause hereditary hyperaldosteronism?

Abstract

The inward rectifier K+ channel subfamily (Kir) consists of seven channels (Kir1–7). Genetic mutations of Kir channels are known to induce hereditary diseases, such as Bartter syndrome (Kir1.1), Anderson syndrome (Kir2.1) and familial hypoglycaemia (Kir6.2). Kir3 is a G-protein coupled inward rectifier K+ channel which is activated by the Gβγ subunit released in response to the stimulation of Gi/o-coupled receptors. It is known to play physiological roles in various cells such as neuronal cells, cardiac muscle cells and adrenal cortex endocrine cells. Some hereditary diseases are also kinked to Kir3 channels. Mutations of Kir3.2 in the ion selectivity filter or in the pore helix are known to cause Keppen-Lubinsky syndrome (Masotti et al. 2015), and those of Kir3.4 (GIRK4) are reported to induce adrenal aldosterone producing adenoma and hyperaldosteronism (Choi et al. 2011). K+ channelsmaintain the resting potential and contribute to the repolarizing phase of the action potential. Hyperaldosteronism can be caused by the hyper-excitability of aldosterone secreting cells in adrenal cortex endocrine cells. How can this hyper-excitability be caused by an abnormality of Kir3.4? One possibility is loss of ion selectivity. If Kir3.4 also allows permeation of Na+, the resting potential cannot be maintained, and Ca2+ influx could evoke hormonal secretion. Another possibility is loss-of-function. If Kir3.4 currents are decreased, the resting potential cannot be maintained. The pathophysiological mechanisms arising from these mutations have not been solved conclusively. They may be, at least partly, due to variation of the induced abnormality depending on the position of the mutation. In this issue of The Journal of Physiology, Shalomov et al. (2022) revisited the pathophysiological mechanisms by intensively characterizing the Kir3.4 mutants. Choi et al. (2011) analyzed the ion selectivity of Kir3.1/Kir3.4 (wt or G151R or L168R or T158A) expressed in HEK293T cells, and beautifully showed that all these mutants allow Na+ to permeate as well. They discussed the loss of K+ selectivity induced depolarization and Ca2+ influx, which caused constitutive aldosterone production and cell proliferation. G151 is located in the selectivity filter, while L168 is in the pore helix behind the selectivity filter and T158 is in the extracellular loop just above the selectivity filter. Considering the location of these amino acid residues, an abnormality due to loss of ion selectivity is both understandable and acceptable. Murthy et al. (2014) characterized new disease associated Kir3.4 mutants R52H and E246K in the cytosolic region of Xenopus oocytes. They showed that the K+ selectivity, rectification and sensitivity to the block by TPN-Q were remarkably reduced. Judging from the position of the mutations, the observed phenotypes were rather unexpected and the mechanism underlying the abnormality remained to be elucidated. Shalomov and co-authors aimed to clarify the pathophysiological mechanisms associated with these mutations and performed a detailed analysis of the R52H, E246K and G247R mutants expressed in Xenopus oocytes and in a human adrenocortical carcinoma cell line (HAC15). They showed that homotetramers of the mutants are non-functional, and that heterotetramers with Kir3.1 are functional with far lower current amplitudes than in Kir3.4wt. They also presented convincing results showing that the ion selectivity and rectification of the mutants are not significantly changed. Furthermore, they showed by confocal imaging and FRET analysis that expression on the plasma membrane and the interaction with Gβγ mutants are reduced. They also showed by single channel recording that the open probability of GRIK1/4R52H is reduced. They observed that G247R responded well to VU0529331, a Kir3.4 channel opener, proposing a potential new therapeutic approach. Taken together, they concluded that the pathophysiological mechanism of Kir3.4 mutants R52H, E246K and G247R in the cytosolic region is due not to loss of ion selectivity, but to reduced function/expression (Shalomov et al. 2022). It is crucial to demonstrate that the Kir3.4 mutations actually evoke an increase in aldosterone release from secreting cells. Shalomov et al. (2022) confirmed expression of the Kir3.4 protein in a human adrenocortical carcinoma cell line (HAC15), and showed that the overexpression of Kir3.4 wt, but not of the Kir3.4 R52H mutant, reduced aldosterone secretion. This is good supporting evidence. It would be an interesting task, at the next stage, to present more direct evidence by a straightforward experiment, e.g. a higher aldosterone secretion in Kir3.4 genome-modified cells than in wt cells. Combining evidence from the former study by Choi et al. (2011) and the present study by Shalomov et al. (2022), it is now clear that there are two types of pathophysiological mechanisms for hereditary hyperaldosteronism associated with Kir3.4 mutations, i.e. loss of ion selectivity caused by pore mutations and reduced function/expression caused by cytosolic mutations. On the same topic of the pathophysiological mechanism of disease associated mutants of Kir3, formation of a secondary permeation pathway besides the selectivity filter pathway in Kir3.2 G156S has been reported as a basis for the abnormal ion selectivity (Chen et al. 2022). The study by Shalomov et al. (2022) reminds us that revisiting the pathophysiological mechanisms of other ion channelopathies might be needed to gain a better understanding.