Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology

Abstract

Between 6-20% of the cellular proteome is under circadian control to tune cell function with cycles of environmental change. For cell viability, and to maintain volume within narrow limits, the osmotic pressure exerted by changes in the soluble proteome must be compensated. The mechanisms and consequences underlying compensation are not known. Here, we show in cultured mammalian cells and in vivo that compensation requires electroneutral active transport of Na+, K+, and Cl− through differential activity of SLC12A family cotransporters. In cardiomyocytes ex vivo and in vivo, compensatory ion fluxes alter their electrical activity at different times of the day. Perturbation of soluble protein abundance has commensurate effects on ion composition and cellular function across the circadian cycle. Thus, circadian regulation of the proteome impacts ion homeostasis with substantial consequences for the physiology of electrically active cells such as cardiomyocytes.

CRYPTOCHROME suppresses the circadian proteome and promotes protein homeostasis

“…To validate this, we measured the K + content of cells across the circadian cycle. Consistent with previous investigations 71 , in WT cells, K + and digitonin-extracted cytosolic protein concentrations exhibited antiphasic circadian rhythms (Figure 4D, Figure S5C), with no significant daily variation in total cellular protein. The same was observed in CKO cells (Figure 4E), but with higher relative amplitudes for soluble protein and K + (Figure 4G, H).…”

“…The same was observed in CKO cells (Figure 4E), but with higher relative amplitudes for soluble protein and K + (Figure 4G, H). Considering previous observations 71 , the higher amplitude cytosolic protein rhythm in CKO cells likely drives the higher amplitude K + rhythms. This is facilitated by increased expression and amplitude of SLC12A transporter activity (Figure 4F), which buffers cellular osmotic potential in response to greater changes in cytosolic macromolecular content over the circadian cycle 71 .…”

“…Interrogation of the proteomics dataset then revealed altered expression levels and increased rhythmic amplitudes of many ion transporters in CKO compared with WT cells (Figure S5A, B). This included several members of the SLC12A family of electroneutral transporters (Figure 4F), that contribute to the maintenance of osmotic homeostasis against variations in cytosolic protein concentration in wild type cells over the circadian cycle 71 . Second, since many proteins are cytoplasmic, we considered that if CRY normally suppresses rhythms in the abundance of many individual proteins that normally peak around the same circadian phase (Figure 2E-H) then the consequence of CRY deletion would be to increase the overall amplitude of daily rhythms in soluble cytosolic protein content.…”

“…Previous work has suggested that compensatory K + transport is a fundamental feature of eukaryotic cell biology allowing cells to accommodate changes in cytosolic macromolecular content whilst maintaining osmotic homeostasis 67, 71 . Our present observations suggest that this occurs independently of CRY-mediated transcriptional feedback repression.…”

SARS-CoV2 Infection and the Importance of Potassium Balance

“…Another 7 of the 69 drugs inhibit protein synthesis (34). Although the mechanism is not known, protein synthesis, and potassium abundance are inversely correlated in systems as diverse as yeast, algae, and mouse fibroblasts (40)(41)(42)(43), such that inhibition of protein synthesis would be expected to result in greater intracellular potassium abundance. A further 17 drugs have been shown to affect osmotic or ion homeostasis.…”