TRPC4 belongs to the subfamily of "canonical" Transient Receptor Potential channels and acts as non-selective Ca²⁺-permeable cation channel involved in the regulation of various cell functions, particularly in smooth muscles and CNS. Three calmodulin-binding sites were identified in the C-terminal region of the full-length channel (TRPC4a), while the shorter form (TRPC4ß) lacks two of them.

Preliminary experiments with bovine TRPC4a channels expressed in HEK293 cells had shown that Ca²⁺-dependency of their activation via G-protein pathway has an inverted bell-shaped with anti- peak around 300 nM of free Ca²⁺. Since ß-form cannot be found in bovine cells in nature, a deletion mutant was constructed from TRPC4a channel by deletion of 84 respective amino acids enclosing two calmodulin-binding sites. At the same conditions newly obtained TRPC4ß channels showed a normal bell-shape (but inverted in comparison with a-form) curve of Ca²⁺-dependency with a maximum located in the same range of Ca²⁺ concentrations.

Further investigations were performed at normal concentration of free cytosolic Ca²⁺ (100 nM). Murine TRPC4s (a- and ß-forms) were stably expressed in HEK293 cell line, activation of TRPC4-current was performed via phospholipase C pathway by intracellular introduction of GTP-g-S. Cells were clamped in whole-cell mode and held at -60 mV. Both a- and ß-channels revealed characteristic double-rectification curves in response to voltage ramps applied from -150 to +200 mV. Currents at +150 and -100 mV were chosen as time-course indicators of the behavior of TRPC4-channels. Time-course of murine TRPC4a-current development differed from that of TRPC4ß. While the first one revealed a peak with consequent return to initial level, the latter was characterized by slow increase to constant level.

Introduction of calmodulin inhibitor ophiobolin A (10 mM) from inside completely abolished the fast peak of activation, peculiar to murine TRPC4a-currents. Interestingly, inhibition of calmodulin in TRPC4ß-expressing cells lead currents to reach finally significantly higher (by 6712%) steady level in comparison with non-treated cells. Thus, murine TRPC4 channels seem to possess two different types of calmodulin-binding sites differing by their ability to respond to calmodulin and providing opposite directions of channels modulation. It should be noted, that shapes of IV-plot stayed unaffected for both TRPC4a and ß channels under ophiobolin A treatment, meaning that abovementioned changes do not affect their permeation and voltage-dependence properties.

It should be concluded, that a- and ß-isoforms of TRPC4-channels not only have different patterns of activation through phospholipase C pathway, but also calcium-calmodulin can modulate their functions in opposite ways.