Functional consequences of the CAPOS mutation E818K of Na+,K+-ATPase

Abstract

The cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome is caused by the single mutation E818K of the ␣3-isoform of Na ؉ ,K ؉ -ATPase. Here, using biochemical and electrophysiological approaches, we examined the functional characteristics of E818K, as well as of E818Q and E818A mutants. We found that these amino acid substitutions reduce the apparent Na ؉ affinity at the cytoplasmic-facing sites of the pump protein and that this effect is more pronounced for the lysine and glutamine substitutions (3-4fold) than for the alanine substitution. The electrophysiological measurements indicated a more conspicuous, ϳ30-fold reduction of apparent Na ؉ affinity for the extracellular-facing sites in the CAPOS mutant, which was related to an accelerated transition between the phosphoenzyme intermediates E 1 P and E 2 P. The apparent affinity for K ؉ activation of the ATPase activity was unaffected by these substitutions, suggesting that primarily the Na ؉ -specific site III is affected. Furthermore, the apparent affinities for ATP and vanadate were WT-like in E818K, indicating a normal E 1 -E 2 equilibrium of the dephosphoenzyme. Proton-leak currents were not increased in E818K. However, the CAPOS mutation caused a weaker voltage dependence of the pumping rate and a stronger inhibition by cytoplasmic K ؉ than the WT enzyme, which together with the reduced Na ؉ affinity of the cytoplasmic-facing sites precluded proper pump activation under physiological conditions. The functional deficiencies could be traced to the participation of Glu-818 in an intricate hydrogen-bonding/salt-bridge network, connecting it to key residues involved in Na ؉ interaction at site III.

ATP1A3-Related Disorders: An Ever-Expanding Clinical Spectrum

“…This variant affect the structure of the C-terminal region. It is presumed that these changes affect propagation of membrane potential along the spiral ganglion neurons ( 41 , 78 ).…”

“…Roenn and colleagues characterized the functional defects of the CAPOS ATP1A3 p.Glu818Lys using a combination of biochemical and electrophysiological measurements, which allowed demonstration of a reduced Na + affinity of the transport sites of the CAPOS mutant in internally as well as externally facing conformations. Consequently, the CAPOS mutant pump may fail to clear the neuron fast enough of the accumulated Na + in relation to action potentials, and this defect might be part of the pathophysiological mechanism ( 41 ).…”

De novo ATP1A3 variants cause polymicrogyria

“…Their expression may be unstable, or they may have stable expression with reduced ATPase activity (4). The CAPOS-associated p.Glu818Lys variant affects Na + /K + ion binding and turnover rates for ATP hydrolysis and pump currents (17). In contrast, the polymicrogyria-associated variants seemed to accumulate at the -subunit-binding site of the -subunit, remote from ion-binding sites (10), where AHC-, RDP-, or CAPOS-associated variants rarely exist.…”

ATP1A2- and ATP1A3-associated early profound epileptic encephalopathy and polymicrogyria

“…We used several of these methods to investigate the functional consequences of 14 mutations (Table 2 and Supplementary Table 1) (Toustrup-Jensen et al, 2014;Roenn et al, 2019). We did not further characterize A3-D609Y, A3-D801N, A3-G893W, and A3-L924P as their impact is already well understood as mentioned above, and A3-F857del because Patient 21 was included in this series at a late stage.…”