Korean J Physiol Pharmacol.  2020 Jan;24(1):101-110. 10.4196/kjpp.2020.24.1.101.

Analysis of interaction between intracellular spermine and transient receptor potential canonical 4 channel: multiple candidate sites of negatively charged amino acids for the inward rectification of transient receptor potential canonical 4

• Kim J
• Moon SH
• Kim T
• Ko J
• Jeon YK
• Shin YC
• Jeon JH
• So I

• KMID: 2466574
• DOI: http://doi.org/10.4196/kjpp.2020.24.1.101

Abstract

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.

Keyword

Polyamines; Spermine; Transient receptor potential channels

MeSH Terms

Amino Acids*
Binding Sites
Calcium
Cytosol
Glutamic Acid
Myocytes, Smooth Muscle
Permeability
Polyamines
Receptors, Muscarinic
Spermine*
Transient Receptor Potential Channels
Amino Acids
Calcium
Glutamic Acid
Polyamines
Receptors, Muscarinic
Spermine
Transient Receptor Potential Channels

Figure

• 

  Fig. 1 Current densities of transient receptor potential canonical 4 (TRPC4) channels with different types of mutations. (A–C) Black dots represent current densities without spermine and red dots represent current densities with 1 mM intracellular spermine. (A) D629A mutation showed I+100mV of 36.13 ± 6.77 pA/pF and 17.48 ± 4.52 pA/pF when spermine was infused in pipette solution. (B) D629A/Δ(720–740) mutation showed I+100mV of 196.65 ± 87.39 pA/pF and 10.51 ± 4.16 pA/pF in the presence of intracellular spermine. (C) D629A/Δ(720–820) mutation showed I+100mV of 80.19 ± 16.45 pA/pF and 7.82 ± 2.05 pA/pF in the presence of intracellular spermine. (D) % inhibition of spermine calculated by current ratio between spermine (+) and spermine (−) groups.

• 

  Fig. 2 Spermine blocks transient receptor potential canonical 4 (TRPC4) with similar degrees regardless of the channel's conductance. (A) Current densities of mouse TRPC4 channels co-expressed with Gαi2 proteins. (B) Normalized conductance curve of mouse TRPC4 channel coexpressed with Gαi2 proteins. Half-maximal voltage was −14.94 mV in the absence of spermine and −32.82 mV in the presence of 1 mM intracellular spermine. (C) Current densities of mouse TRPC4 channels activated by 100 nM of extracellular (−)-Englerin A. (D) Normalized conductance curve of mouse TRPC4 channel activated by 100 nM of extracellular (−)-Englerin A. Half-maximal voltage was −0.49 mV in the absence of spermine and −16.8 mV in the presence of intracellular spermine. For (A–D), black dots/lines represent current densities/Boltzmann fit without spermine and red dots/lines represent current densities/Boltzmann fit with intracellular spermine. Gray lines show normalized conductance of each group. (E) Current densities of each activation system at +100 mV and −100 mV. (F) Magnitude of I+100mV inhibition by spermine with respect to maximum I+100mV at each activation group.

• 

  Fig. 3 Glutamate 649th and 687th are responsible for intracellular spermine binding. (A–E) Current densities of mutant channels. (A) D629A/E648A/Δ(720–740), (B) D629A/E649A/Δ(720–740), (C) D629A/E687A/Δ(720–740), (D) D629A/E698A/Δ(720–740), (E) D629A/E708A/Δ(720–740). (F) % inhibition of spermine calculated by current ratio between 1 mM spermine (+) and spermine (−) groups.

• 

  Fig. 4 Amino acid notations based on Cryo-EM structure of mouse transient receptor potential canonical 4 (TRPC4) channel. (A) D629, (B) D632, (C) E648, (D) E649, (E) residues from 720-740. For visual preferences, subunits in front of and behind of the screen was omitted on purpose. Each residue was colored red.

• 

  Fig. 5 Structure of tetrameric transient receptor potential canonical 4 (TRPC4) and its putative spermine binding sites. (A) Upper panel; residues in Fig. 4 was all highlighted with red sticks. Yellow helices represent pore-helices (504–626), green helices represent TRP helix (634–658) and cyan helices represent C-terminal helix (696–753). Lower panel; surface potential of TRPC4 channel. For detailed information regarding calculation, please see Methods. Local potential minimum created by E648, E649 are highlighted with black arrow. (B) Model for blocking action of intracellular spermine in inwardly rectifying potassium channel (IRK) and TRPC4 channel.

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