Intracellular nucleotides and polyamines inhibit the Ca 2+ -activated cation channel TRPM4b

Abstract

TRPM4b (in contrast to the short splice variant TRPM4a) is a Ca(2+)-activated but Ca(2+)-impermeable cation channel. We have studied TRPM4 currents in inside-out patches. Supramicromolar Ca(2+) concentrations applied at the inner side, [Ca(2+)](i), activated TRPM4 with an EC(50) value of 0.37 mM, a value that is much higher than that of whole-cell currents. Current amplitudes decreased above 1 mM [Ca(2+)](i), (IC(50) 9.3 mM). Sr(2+) but not Ba(2+)could partially substitute for Ca(2+). ATP, ADP, AMP and AMP-PNP all quickly and reversibly inhibited TRPM4 with IC(50) values between 2 and 19 microM (at +100 mV). Adenosine also blocked TRPM4 at 630 microM. The block at high ATP concentrations was incomplete and was not affected by the presence of free Mg(2+). ADP induced the most sensitive block with an IC(50) of 2.2 microM. For inhibition of TRPM4 by free ATP(4-), an IC(50) value of 1.7+/-0.3 microM was calculated. GTP, UTP and CTP at concentrations up to 1 mM did not induce a similar block. Spermine blocked TRPM4 currents with an IC(50) of 61 microM. In conclusion, TRPM4 is a channel that can be effectively modulated by intracellular nucleotides and polyamines.

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

“…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.…”

“…Because the ATP specificity in TRPM4 is mainly defined by the adenine moiety of the nucleotide (Fig. 2c), ADP and AMP can also bind and inhibit TRPM4 15 . The basic residues surrounding the ATP site likely stabilize the negatively charged triphosphate group and may prevent the binding of ATP-Mg.…”

“…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 .…”

On Potential Interactions between Non-selective Cation Channel TRPM4 and Sulfonylurea Receptor SUR1

“…4A). After desensitization, we tested the effect of 10 M free ATP, as reported (10). Steady-state currents were significantly inhibited by ATP (Fig.…”

“…These channels are structurally distant from the Kir family, but intriguingly, TRPM4, similar to Kir6.2, is sensitive to inhibition by cytoplasmic ATP in the micromolar range (10). It has been reported that, after traumatic brain or spinal cord injury, or ischemia after stroke, SUR1 is up-regulated in neurovascular cells such as reactive astrocytes and endothelial cells at the site of injury (11,12).…”