Disease-linked mutations alter the stoichiometries of HCN-KCNE2 complexes

Abstract

The four hyperpolarization-activated cylic-nucleotide gated (HCN) channel isoforms and their auxiliary subunit KCNE2 are important in the regulation of peripheral and central neuronal firing and the heartbeat. Disruption of their normal function has been implicated in cardiac arrhythmias, peripheral pain, and epilepsy. However, molecular details of the HCN-KCNE2 complexes are unknown. Using single-molecule subunit counting, we determined that the number of KCNE2 subunits in complex with the pore-forming subunits of human HCN channels differs with each HCN isoform and is dynamic with respect to concentration. These interactions can be altered by KCNE2 gene-variants with functional implications. The results provide an additional consideration necessary to understand heart rhythm, pain, and epileptic disorders.

Disturbed microbial ecology in Alzheimer’s disease: evidence from the gut microbiota and fecal metabolome

“…Several studies suggest that GMB impact brain through a bidirectional communication system that is connected via neural, immune, endocrine, and metabolic pathways [ 17 – 20 ]. Although 16S ribosomal RNA (rRNA) gene sequencing allows for profiling of complex microbial composition and indicating the microbial entities [ 21 ], annotation and the actual microbial activity of GMB is sparse as 16S rRNA gene sequence only provides a global composition analysis but cannot distinguish alive and dead microbes [ 22 ]. Fecal metabolites, served as the metabolic output of both GMB and cellular metabolism occurring inside the human intestinal tract, could complement sequencing-based approaches with showing functional readout of microbial activity [ 23 ].…”

Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry

“…In addition, using photobleaching, the Felipe lab recently counted 4:4 for KCNA3:KCNE4, although they too reported a variable KCNE4 stoichiometry in the complexes depending on the relative expression level, and concluded that functional attributes varied with stoichiometry 78 . Finally, using photobleaching, the D’Avanzo group concluded that complexes formed by pacemaker (HCN) channel alpha subunit and KCNE2, which may contribute to pacemaking in the heart and/or brain, have an expression-level dependent variable stoichiometry of between 4:1 and 4:4 (HCN:KCNE2) 79 .…”

Determination of oligomeric states of proteins via dual-color colocalization with single molecule localization microscopy

“…Stepwise photobleaching relies on the irreversible and stochastic photobleaching of fluorescent tags linked to the subunit to be counted, and the resulting intensity steps depend on the number of subunits within the complex being tested. Such data can be acquired using comparatively inexpensive microscopes which has undoubtedly led to the widespread adoption of this method (1,6,9,10).…”

“…Stepwise photobleaching relies on the irreversible and stochastic photobleaching of fluorescent tags linked to the subunit to be counted, and the resulting intensity steps depend on the number of subunits within the complex being tested. Such data can be acquired using comparatively inexpensive microscopes which has undoubtedly led to the widespread adoption of this method (Bartoi et al, 2014; Coste et al, 2012; Lussier et al, 2019; Ulbrich & Isacoff, 2007). However, this approach is only feasible for low expression densities as it is limited by the resolution of light microscopy.…”