Background
Voltage-gated sodium channels (NaV) are crucial transmembrane proteins composed of a pore-forming α-subunit and auxiliary β-subunits. Due to their role in the generation and propagation of action potentials, many venoms have evolved to target these subunits, causing symptoms such as pain or incapacitation. However, recent research has identified an alternative mechanism for the modulation of the NaV complex through the accessory protein TMEM233. To date, the toxins from the Dendrocnide family have been the only toxins identified to modulate NaV through TMEM233, altering its activation and fast inactivation properties. Specifically, excelsatoxin A (ExTxA), originating from the Giant Australian Stinging Tree (Dendrocnide excelsa), has been used as a tool to probe TMEM233.
Alongside its expression in the peripheral nervous system, TMEM233 has been found to be highly enriched in fast-twitch skeletal muscle. Previous research has demonstrated the role of TMEM233 (re-termed muscle-enriched calcium regulator; MCARE) in sarco/endoplasmic reticulum calcium reuptake, regulated by the Ca2+-ATPase 1 pump (SERCA1), however, its role on plasma membrane associated signalling in skeletal muscle remains unknown.
Methods
Immunofluorescence imaging was used to localise TMEM233 in wild-type quadriceps muscle sections stained with ExTxA-488, Caveolin-3 and DAPI. ExTxA-488 enabled the localisation of TMEM233 through toxin binding, while Caveolin-3 served as a marker for skeletal muscle plasma membrane (sarcolemma). In addition to this, fluorescence-based imaging was used to investigate the role of TMEM233 in myofibres isolated from the flexor digitorum brevis. Using ExTxA as a probe, concentration responses were measured in the presence and absence of extracellular Ca2+.
Results
Immunofluorescent imaging of ExTxA-488, Caveolin-3 and DAPI revealed partial overlap between the markers, suggesting the presence of TMEM233 on the sarcolemma. Additionally, ExTxA produced concentration dependent increases in calcium release at nanomolar concentrations. Chelating extracellular Ca2+ abolished this response, indicating that ExTxA causes calcium signalling dependent on extracellular calcium influx rather than release from intracellular stores alone.
Conclusion
The partial co-localisation of ExTxA-488 with Caveolin-3 provides evidence for TMEM233 expression on the sarcolemma. Together with the dependence of ExTxA induced calcium responses on extracellular Ca²⁺, these findings support a potential role for TMEM233 in plasma membrane-associated signalling in skeletal muscle.