Programme Contents

pH dependence of the Ca²⁺ release activated Ca²⁺ (CRAC) channel

CRAC channels activated by the depletion of intracellular Ca²⁺ stores provide a major pathway for Ca²⁺ entry in many cell types. Characteristic properties of CRAC channels include high selectivity for Ca²⁺ over monovalent cations, feedback inhibition by permeating Ca²⁺, known as fast Ca²⁺ dependent inactivation (FCDI), and block by low external pH (Malayev & Nelson, 1995). The functional CRAC channels are composed of a tetramer of the Orai1 proteins, which forms the channel pore, and a protein called stromal interaction molecule 1 (STIM1), a Ca²⁺ binding protein that plays the role of Ca²⁺ sensor in the endoplasmic reticulum (Soboloff et al., 2006). The glutamate 106 residue (E106) in a predicted transmembrane domain of Orai1 has been reported to act as the selectivity filter and to play a role in FCDI of CRAC channels (Yamashita et al., 2008). In this work we show that glutamate 106 is also a protonation site responsible for I_CRAC block at low pH.

STIM1 and Orai1 were previously subcloned into pCMV-Sport6 and the GFP co-expressing vector pAdTrack-CMV (Scrimgeour et al., 2009). The Orai1 E106D mutation was generated using pCMV-Sport6-Orai1 as a template according to the protocol specified by the QuikChange II site-directed mutagenesis kit (Stratagene). Whole-cell patch clamping was performed at room temperature using a computer based patch-clamp amplifier (EPC-9, HEKA Elektronik) and PULSE software (HEKA Elektronik).

I_CRAC mediated by heterologously expressed Orai1 and STIM1 was inhibited by low pH reaching virtually complete block at pH 5.5. The apparent pKa of CRAC channel pH dependence was 7.8±0.1 (n=4). The E106D Orai1 mutant, which has higher selectivity for Na⁺ over Ca²⁺ and is blocked by Ca²⁺ in time and voltage dependent manner (Yamashita et al., 2008), showed no such dependence on pH. In contrast, lowering pH from 7.4 to 6.3 or below increased the amplitude of the current and reduced the extent of inactivation at negative potentials suggesting that the Ca²⁺ block of Na⁺ current was reduced. The apparent pKa of the block of Na⁺ conductance through E106D mutant by Ca²⁺ was 6.1±0.1 (n=4). Investigation of Ca²⁺ currents mediated by this mutant in the absence of all permeable monovalent cations in the external solution revealed that FCDI of E106D is much faster than that of WT Orai1 and is progressively reduced at lower external pH.

Overall, these results suggest that ring of negative charges at position 106 in the Orai1 pore controls not only the selectivity of the channel, but also contributes to a complex mechanism of FCDI and accounts for the pH dependence of CRAC channel.

Malayev A & Nelson DJ (1995). Journal of Membrane Biology 146, 101-111.

Scrimgeour N, Litjens T, Ma L, Barritt GJ & Rychkov GY (2009). Journal of Physiology 587, 2903-2918.

Soboloff J, Spassova MA, Tang XD, Hewavitharana T, Xu W & Gill DL (2006). Journal of Biological Chemistry 281, 20661-20665.

Yamashita M, Navarro-Borelly L, McNally BA & Prakriya M (2007). Journal of General Physiology 130, 525-540.