Abstract
TRPM4, a Ca(2+)-activated cation channel of the transient receptor potential superfamily, undergoes a fast desensitization to Ca(2+). The mechanisms underlying the alterations in Ca(2+) sensitivity are unknown. Here we show that cytoplasmic ATP reversed Ca(2+) sensitivity after desensitization, whereas mutations to putative ATP binding sites resulted in faster and more complete desensitization. Phorbol ester-induced activation of protein kinase C (PKC) increased the Ca(2+) sensitivity of wild-type TRPM4 but not of two mutants mutated at putative PKC phosphorylation sites. Overexpression of a calmodulin mutant unable to bind Ca(2+) dramatically reduced TRPM4 activation. We identified five Ca(2+)-calmodulin binding sites in TRPM4 and showed that deletion of any of the three C-terminal sites strongly impaired current activation by reducing Ca(2+) sensitivity and shifting the voltage dependence of activation to very positive potentials. Thus, the Ca(2+) sensitivity of TRPM4 is regulated by ATP, PKC-dependent phosphorylation, and calmodulin binding at the C terminus.
Highlights
TRPM4 1 is a Ca2ϩ-activated and voltage-dependent Ca2ϩimpermeable cation channel with a unitary conductance of 25 picosiemens that belongs to the melastatin subfamily of transient receptor potential membrane proteins [1,2,3,4]
The Ca2؉ sensitivity of TRPM4 is regulated by ATP, protein kinase C (PKC)-dependent phosphorylation, and calmodulin binding at the C terminus
We report that the Ca2ϩ sensitivity of TRPM4 is regulated by cytosolic ATP, protein kinase C (PKC)-dependent phosphorylation, and calmodulin (CaM), and we identify distinct residues in the channel protein that are crucial for the modulation by these cellular factors
Summary
TRPM4 1 is a Ca2ϩ-activated and voltage-dependent Ca2ϩimpermeable cation channel with a unitary conductance of 25 picosiemens that belongs to the melastatin subfamily of transient receptor potential membrane proteins [1,2,3,4]. Recent studies on TRPM4 exhibit an unusually large variability in reported values for Ca2ϩ sensitivity and activation time courses [1,2,3,4]. Most likely, these discrepancies reflect a highly regulated Ca2ϩ affinity of TRPM4, which may be of physiological relevance. We report that the Ca2ϩ sensitivity of TRPM4 is regulated by cytosolic ATP, protein kinase C (PKC)-dependent phosphorylation, and calmodulin (CaM), and we identify distinct residues in the channel protein that are crucial for the modulation by these cellular factors
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