Background
The cellular response to hypoxia plays a crucial role in ischemic heart disease. It determines cell survival after ischemia-reperfusion and contributes to the associated risk of developing life-threatening arrhythmias. Recently, TRPM4 ion channel activity has been identified as a major factor in the pathology of arrhythmia, conduction block and ischemia-reperfusion. We now present data that suggest a novel mechanism of TRPM4 activation in hypoxia, involving the scaffolding protein caveolin and the small ubiquitin-like modifier protein SUMO.

Results
We have found broadly overlapping expression of TRPM4 and caveolin at the plasma membrane of freshly dissociated murine cardiomyocytes and our co-immunopurification experiments in a heterologous expression cell line indicate that both proteins can form complexes with each other. When we overexpressed the SUMOylation enzymes Ubc9 and PIAS3 in the same cells, caveolin and TRPM4 protein complexes dissociated, while TRPM4 current density was greatly increased. We also tested if the cell surface expression of TRPM4 was changed by the overexpression of Ubc9 and PIAS3 and found no differences. The TRPM4-R164W mutation has been reported as insensitive to SUMOylation effects. We created recombinant knock-in cell lines of both wild type and of R164W mutant TRPM4 genes for stable heterologous expression, then measured current density in response to increased SUMOylation activity. TRPM4 R164W current density was substantially increased compared to wild type TRPM4 but did not increase further with Ubc9/PIAS3 overexpression. TRPM4-R164W protein surface expression was the same as for wild type. However, we found significantly reduced caveolin binding of TRPM4-R164W channels compared to wild type in co-IP experiments. We then reduced TRPM4-caveolin binding by targeted mutation of a putative caveolin binding motif within TRPM4, which also caused an increase of TRPM4 current density compared to wild type.

Discussion
Our data indicate that cell surface TRPM4 channels are reversibly inactivated by binding to caveolin. This binding is substantially reduced by upregulation of SUMOylation enzymes, resulting in TRPM4 activation. The SUMOylation system is strongly activated under hypoxic conditions through upregulation of SUMO-1 and of the SUMOylation enhancer RSUME. We therefore propose SUMOylation-regulated Caveolin-TRPM4 complex destabilization may be the molecular mechanism that provides the missing link between hypoxia and the pro-arrhythmogenic and conduction-slowing effects of the TRPM4 channel (see schematic).  Low oxygen availability induces RSUME expression, which amplifies cellular SUMOylation through direct binding of the E2-ligase Ubc9. TRPM4-caveolin complexes, in which TRPM4 is not active, are subsequently destabilized, either by SUMOylation of TRPM4, of caveolins or of other factors. Release from caveolin binding then results in TRPM4 activation.

Ongoing project
We now investigate the molecular interaction between TRPM4 and caveolin, as well as the upstream mechanism of SUMOylation-linked TRPM4 activation during hypoxia. While switching our focus to primary cardiomyocytes and whole animal experiments, we also expand our experimental possibilities with the generation of a TRPM4-R164W mouse model.