Negatively Charged Amino Acids Near and in Transient Receptor Potential (TRP) Domain of TRPM4 Channel Are One Determinant of Its Ca2+ Sensitivity.

Results: Cobalt potentiated activation of TRPM4 by Ca 2+ . Mutations of negatively charged amino acids in TRP domain reduced Ca 2+ sensitivity. Conclusion: The acidic amino acids are required for the proper activation of TRPM4 by Ca 2+ . Significance: A novel role of TRP domain in TRPM4 suggested. ABSTRACT Transient Receptor Potential (TRP) channel Melastatin subfamily member 4 (TRPM4) is a broadly expressed nonselective monovalent cation channel. activated by membrane depolarization and intracellular Ca 2+ , which is essential for the activation. The Ca 2+ sensitivity is known

For example, the EC 50 for Ca 2+ after desensitization was reported to be 520 μM, and that after application of PIP 2 was 120 μM (16).
Positively charged amino acids in a C-terminal pleckstrin homology (PH) domain were identified as important determinants of PIP 2 action (17).
However, the mechanism of how PIP 2 increases the Ca 2+ sensitivity of TRPM4 has not been revealed.
where G max is fixed to the normalized conductance at +100 mV, V m is the membrane potential, V 1/2 is the membrane potential at which the conductance is half of G max , dx is the slope factor, and f is the voltage-independent conductance fraction. All curves were obtained by fitting the data averages.

Effects of divalent cations on TRPM4 currents
Rat TRPM4 showed outward rectifying currents after the inside-out patch excision in the presence of 1 mM Ca 2+ in the perfusate, and the currents were decreased probably mainly by a decline in the sensitivity of the channels to Ca 2+ (14), which is most likely due to the loss of PIP 2 as in well-studied human TRPM4 (16,17,29) (Fig. 1A).

Mutations of negatively-charged amino acids near and in TRP domain reduce Ca 2+ sensitivity and voltage dependence
To explore the divalent cation binding sites of   5B). As shown in Figure Fig. 8F and Table 1) but hardly affected the EC 50 for Co 2+ (WT: Fig. 8E and Table 1).      D1049N, E1062A, and E1062Q). EC 50 and Hill coefficients for Ca 2+ were determined without additional conditions or also in the presence of 1 mM Co 2+ or 30 μM diC8-PI(4,5)P 2 (PIP 2 ). EC 50 and Hill coefficients for Co 2+ were calculated from the decrease of EC 50 for Ca 2+ by the simultaneous application of Co 2+ , shown in Fig. 8E. The EC 50 and Hill coefficients for PIP 2 were evaluated in the presence of Ca 2+ , shown in Fig. 9D. The Ca 2+ concentrations used for wild-type (WT) TRPM4, D1049N, and E1062Q were 100 µM, 300 µM, and 1 mM Ca 2+ , respectively. The ratios of the EC 50 of mutants to that of WT TRPM4 are shown in the parentheses. The current amplitudes at +100 mV were used for the calculation.   were normalized to the current amplitude recorded in the presence of 1 mM Ca 2+ right before exposure to the FA or 1 mM Co 2+ . FA inhibited TRPM4 currents significantly regardless of whether TRPM4 currents were potentiated by Co 2+ . *: P < 0.05, **: P < 0.01. n = 4 or 6.  suggested that there are at least two divalent cation binding sites in TRPM4. The 1st binding site is sensitive to Ca 2+ but insensitive to Co 2+ , Mn 2+ , and Ni 2+ . Accordingly, it is relatively Ca 2+ -specific. The 2nd binding site is sensitive to Co 2+ , Mn 2+ , and Ni 2+ .                Figure 9, Yamaguchi, et al.