Subunit stoichiometry and channel pore structure of ion channels: all for one, or one for one?

Ion channels are macromolecular protein complexes that open and close their pores to control rapid ion fluxes across the cell membranes (Hille, 2001). In general, a single ion-conducting pore is usually formed at the central axis by symmetrical assembly of homologous structural units, either in multimeric pore-forming subunits or internal repeats of a single pore-forming polypeptide. Based on the stoichiometry of structural units surrounding the channel pore (Unwin, 1989; Hille, 2001), most known ion channels can be categorized into classes of three, four, five and six structural units (Fig. 1A). Recent biochemical analysis and atomic force microscopy imaging suggest that P2X receptors contain three subunits. The voltage-gated Na+, K+ and Ca2+ channels, inositol 1,4,5-trisphosphate receptor and ryanodine receptor Ca2+ release channels, HCN channels, CNG channels, TRP channels, and ionotropic glutamate receptors share the four-unit stoichiometry. Some ligand-gated receptors such as nicotinic acetylcholine receptors, GABAA receptors and glycine receptors belong to the five-unit class, while the gap junction channels fit into the six-unit class. Figure 1 Schematic representations of stoichiometry compositions of ion channel pore-forming structural units In contrast, a few types of multimeric channels possess multiple ion-conducting pores, with each monomeric subunit containing a functionally independent channel pore (Fig. 1B). For instance, the dimeric CLC Cl− channels acquire two pores while the tetrameric aquaporin channels contain four water pores. Pore structure plays a critical role in determining the selective permeability of each type of ion channel. Therefore, to understand the functional properties and physiological significance of any channel protein, it is crucial to determine the stoichiometry of pore-forming subunits and the extent and nature of subunit interactions. Ca2+ release-activated Ca2+ (CRAC) channels mediate highly Ca2+ selective, store-operated currents in many cell types including lymphocytes. Recent genetic linkage analysis and genome-wide RNAi screens as well as subsequent mutagenesis studies have elegantly revealed that Orai1 or CRACM1, a four-transmembrane spanning plasma membrane protein, is the pore-forming subunit of the CRAC channel (Feske, 2007). Orai1 and its two closely related Orai family members, Orai2 and Orai3, can assemble into homo- and hetero-multimers (Lis et al. 2007). However, little is known about the stoichiometry of Orai1 subunits in forming a functional CRAC channel – does the composition of a CRAC channel fall into the common three-, four-, five-, or six-structural-units class? This question is particularly intriguing since Orai1 has no obvious channel-like domains conserved in many other types of plasma membrane ion channels and CRAC channels exhibit distinct biophysical properties and store-operated characteristics. In a recent report published in The Journal of Physiology, Shuttleworth and colleagues (Mignen et al. 2007) addressed this question by using cDNA constructs encoding tandem fusions of two to four Orai1 subunits in a single polypeptide, an approach similar to that employed in defining the subunit stoichiometry of the voltage-gated K+ channel (Liman et al. 1992). Orai tandem constructs were expressed in HEK293 cells stably expressing STIM1, a Ca2+ store sensor protein involved in store-operated Ca2+ entry. Instead of using a channel mutant with altered sensitivity for a channel blocker (Liman et al. 1992), the authors took advantage of the dominant-negative E106Q Orai1 mutant, which significantly inhibits both endogenous ICRAC in HEK293 cells and enormously increased ICRAC as a result of coexpression of wild-type Orai1 and STIM1. Consistent with previous reports (Feske, 2007), the authors show that coexpression of the E106Q mutant essentially eliminates enhanced ICRAC in HEK293 cells stably expressing both STIM1 and Orai1 monomers even though both WT and mutant Orai1 molecules target to the plasma membrane normally (their Fig. 1A). Therefore, Orai1 monomers are unlikely to be able to form functional CRAC channel pores by themselves alone and multimerization of Orai1 monomers is required for CRAC channel function. So what is the nature of subunit stoichiometry for a functional CRAC channel? The authors tested the inhibitory effect of the E106Q mutant on the capability of tandem Orai1 constructs encoding two to four linked Orai monomers in inducing macroscopic ICRAC in HEK293 cells stably expressing STIM1. Their working models (their Fig. 2) are based on the hypothesis that the E106Q mutant will diminish or completely abolish the augmented ICRAC only when coexpressed with tandem Orai1 constructs encoding fewer subunits than required for the formation of a functional channel pore. If the tandem Orai1 construct can form a functional channel pore by itself, addition of E106Q mutant will have little effect on the macroscopic ICRAC. Indeed, the authors demonstrated that coexpression of the monomeric E106Q mutant totally eliminated ICRAC induced by the trimeric Orai1 construct but without any obvious inhibitory effect of the tetrameric Orai construct (their Figs 3 and 4). The dimeric Orai1 constructs showed partial inhibition by the E106Q mutant, suggesting that dimeric Orai1 could further assemble into functional CRAC channels by itself, but the E106Q mutant might also be incorporated with the dimeric Orai1. All this evidence converges to suggest that the tetrameric Orai1 construct constitute a functional CRAC channel pore. However, is it possible that CRAC channels, like the CLC Cl− channel and the aquaporin channel, contain more than one channel pore in a tetramer, i.e. two parallel dimers? This notion is very appealing since two transmembrane segments (TMS) of Orai1, TMS1 and TMS3, form an intragenic internal repeat (Cai, 2007). TMS1 and TMS3 of Orai1 have been shown to contribute to the Ca2+ selectivity of the CRAC channel pore (Feske, 2007). In tetrameric aquaporins, two tandem repeats of a single subunit are sufficient to form a functionally independent water channel pore (Verkman, 2005). To address this question, the authors introduced a single E106Q mutation into the Orai1 tetrameric construct to form a WT–WT–WT–E106Q tetramer. If the Orai1 tetramer forms two parallel dimers and/or comprises more than one channel pore (two or four), the single E106Q mutation will likely inactivate only one of the channel pores and render the other channel pores functionally intact (unless these channel pores are somehow functionally interconnected). Indeed, this tetramer construct with a single E106Q mutation did not generate any obvious ICRAC above the background currents recorded in the control cells. Therefore, it is unlikely that the Orai1 tetramer is composed of two functional dimeric channels and possesses more than one channel pore. In conclusion, Mignen et al. (2007) provided compelling initial evidence to demonstrate the tetrameric subunit composition of functional CRAC channels. Like the majority of other four-subunit class channels, four Orai1 subunits possibly assemble symmetrically to form a CRAC channel pore at the central axis. Since functional coupling between STIM1 and Orai1 is required for CRAC channel activation, it will also be interesting to determine the stoichiometry of STIM1. STIM1 undergoes oligomerization in response to store depletion, before redistributing at distinct membrane regions. If a functional CRAC channel is composed of four Orai1 subunits, how many STIM1 monomers are present in the STIM1 oligomer? In the skeletal type excitation–contraction coupling, four skeletal muscle DHPR α1 subunits are believed to physically associate with four subunits of the skeletal muscle ryanodine receptor, RyR1. Thus, it is possible that STIM1 tetramers might also be formed to activate tetrameric CRAC channels. In addition, Orai2 and Orai3 diverged from Orai1 at different evolutionary stages with distinct characteristics (Cai, 2007). Identification of the subunit stoichiometry of CRAC channels will also help further investigation of biochemical and biophysical properties of different Orai hetero-multimers (Lis et al. 2007), and, therefore, help define the compositions of endogenous CRAC channels in native cell types. This elegant study by Shuttleworth and colleagues (Mignen et al. 2007) may serve as a useful guide for future molecular, biophysical, and biochemical analyses of function and mechanism for CRAC channels.