Molecular determinant of sensing extracellular pH in classical transient receptor potential channel 5 Min Ji Kim a , Jae-Pyo Jeon a , Hyun Jin Kim a , Byung Joo Kim a , Young Mee Lee b , Han Choe c , Ju-Hong Jeon a , Seon Jeong Kim d , Insuk So a, * a Center for Bio-Artificial Muscle and Department of Physiology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Republic of Korea b Department of Dermatology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Republic of Korea c Department of Physiology, University of Ulsan College of Medicine, Republic of Korea d Center for Bio-Artificial Muscle, Hanyang University, Seoul 133-791, Republic of Korea Received 17 October 2007 Available online 5 November 2007 Abstract The classical transient receptor potential channel 5 (TRPC5) is a molecular candidate for nonselective cation channel (NSCC) acti- vated by muscarinic receptor stimulation whereas extracellular pH inhibits or enhances NSCC activated by muscarinic receptor stimu- lation depending on extracellular cation compositions in native tissues. We investigated the effect of extracellular pH on TRPC5 and determined amino acid residues responsible for sensing extracellular pH. Extracellular acidosis inhibits TRPC5 with pK a of 6.24. Under 50 mM intracellular HEPES buffer condition, extracellular acidosis inhibits TRPC5 with pK a of 5.40. We changed titratable amino acids (C, D, E, H, K, R, Y) to nontitratable amino acids (A, N, Q, N, N, N, F) within pore region between transmembrane segments 5 and 6 in order to determine the residues sensing extracellular pH. Glutamate (at the position 543, 595, and 598), aspartate (at the position 548) and lysine (at the position 554) were responsible for sensing extracellular pH. The effect of extracellular pH in TRPC5 was also dependent on the composition of extracellular monovalent cations. In conclusion, TRPC5 is a molecular candidate for NSCC activated by musca- rinic receptor stimulation, has glutamate amino acid residues responsible for sensing extracellular pH, and has a unique gating property depending on the composition of extracellular monovalent cations. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Transient receptor potential channel; Extracellular pH; Nonselective cation channel; Muscarinic receptor Extracellular pH modulates the pattern and amplitude of smooth muscle contractions associated with action potential, high K + , or acetylcholine. Extracellular acidosis inhibits smooth muscle contraction by modulating trans- membrane calcium influx, receptor sensitivity and actomy- osin ATPase. Nonselective cation channel activated by muscarinic receptor stimulation is involved in calcium homeostasis by inducing membrane depolarization and cal- cium influx through channel itself in visceral smooth mus- cles [1]. Initially Inoue et al. showed that extracellular acidosis enhanced NSCC activated by muscarinic receptor stimulation [2]. In subsequent study, however, Zholos and Bolton argued that the effect of extracellular acidosis on NSCC depended on the compositions of extracellular monovalent cations [3]. Under symmetrical Cs condition (140 mM extracellular Cs and intracellular Cs), extracellu- lar acidosis inhibited NSCC activated by muscarinic recep- tor stimulation, whereas extracellular acidosis increased NSCC activated by muscarinic receptor stimulation under the normal Tyrode condition [3]. Previously we showed that TRPC5 is a molecular candi- date for NSCC activated by muscarinic receptor stimula- tion [4,5]. In the following studies, we showed that TRPC5 has two regulatory pathways as NSCC acti- vated by muscarinic receptor stimulation in native tissues 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.10.154 * Corresponding author. Fax: +82 2 763 9667. E-mail address: insuk@plaza.snu.ac.kr (I. So). www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 365 (2008) 239–245