Regulation of the Ca2+ Sensitivity of the Nonselective Cation Channel TRPM4

Abstract

TRPM4, a Ca2؉ -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 wildtype 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. TRPM41 is a Ca 2ϩ -activated and voltage-dependent Ca 2ϩ -impermeable cation channel with a unitary conductance of 25 picosiemens that belongs to the melastatin subfamily of transient receptor potential membrane proteins (1-4). Ca 2ϩ -activated, Ca 2ϩ -impermeable nonselective cation channels that share functional properties with expressed TRPM4 (or the closest homologue, TRPM5) have been found in many excitable and non-excitable cells (Refs. 5-8; for reviews, see Refs. 9 and 10). These nonselective channels may regulate important processes including cardiac rhythmicity and neural bursting activity, and their Ca 2ϩ -dependent activation has been suggested to form a general feedback control mechanism for Ca 2ϩ influx in nonexcitable cells.The functional analysis of TRPM4 and TRPM5 and their comparison with native nonselective cation channels are complicated by a peculiar property of these channels: when activated by an increase in free intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ), the currents decay rapidly due to a decrease in the sensitivity of the channels to Ca 2ϩ (1,3,4,11). Moreover, recent studies on TRPM4 exhibit an unusually large variability in reported values for Ca 2ϩ sensitivity and activation time courses (1-4). Most likely, these discrepancies reflect a highly regulated Ca 2ϩ affinity of TRPM4, which may be of physiological relevance.Another puzzling property of TRPM4 is its sensitivity to ATP. We have shown recently that cytosolic ATP 4Ϫ acts as a potent inhibitor of TRPM4 currents in inside-out patches, with half-maximal inhibition at ϳ2 M (3). However, robust TRPM4 currents can be measured in the whole-cell mode, even under conditions in which the free cytosolic ATP 4Ϫ concentration exceeds 100 M. One possible explanation of this apparent paradox could be that ATP has both an inhibitory and a stimulatory effect on TRPM4, but experimental data to support this idea are currently lacking.In the present study, we investigated potential cellular...

Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel TRPM4

“…Only TRPM4 is expressed in BMMCs, and the lack of TRPM4 in Trpm4 -/-mast cells was not compensated for by upregulation of TRPM5 expression. The properties of the endogenous CAN channel in BMMCs described here are very similar to the properties of TRPM4 currents recorded after overexpression of Trpm4 cDNA in HEK293 cells 16,17,24,36 . Both currents had identical permeability profiles and had similar single-channel conductance.…”

Structures of the calcium-activated, non-selective cation channel TRPM4

“…The previously proposed modulation sites for phosphorylation, PtdInsP 2 binding or calmodulin binding at the C-terminal domain do not appear to be accessible based on the structure 14,16 . However, the structural feature of the C-terminal domain would imply that the coiled coil is capable of undergoing an up-and-down sliding motion, which, in turn, can induce lateral movement through the stretcher helices and propagate to the channel transmembrane pore through the middle tier LHD (Fig.…”

“…The 400-residue NBD, hypothesized to be the ATP binding site 16 , is an α/β protein with a central β-sheet formed by nine predominantly parallel β-strands (β3-β11) and its overall architecture partially resembles the Rossmann-fold dinucleotide binding proteins (Fig. 2a).…”

“…Cytosolic ATP has divergent effects on TRPM4 activity: free ATP but not the Mg 2+ -chelated ATP (ATP-Mg) can inhibit the channel 15 , while exposing desensitized TRPM4 to ATP-Mg can alleviate desensitization, thereby increase channel conductance 13,16 . To define the role of ATP binding at NBD, we mutated the three aromatic residues at the ATP binding site to alanine, individually.…”

“…While PtdInsP 2 alone cannot activate the channel, its presence with Ca 2+ reverses channel desensitization, potentiates Ca 2+ sensitivity, and mitigates voltage dependence of the channel 13,14 . Cytosolic ATP can modulate TRPM4 activity in a seemingly paradoxical manner – it inhibits TRPM4 current but can also reverse Ca 2+ desensitization 13,15,16 .…”