Functional TRPM (transient receptor potential melastatin) ion channels are multimers, thought to be tetramers. Subunit interaction is the prerequisite step in channel assembly, and the specificity of subunit interaction is crucial in assembling channels with distinct functional properties. In addition, expression of short non-functional subunits and their interaction with full-length subunits serve as one of the post-translational mechanisms regulating the channel activity. This paper aims to provide an overview of the current knowledge of TRPM subunit interactions and their roles in assembly and functional regulation of the TRPM channels.
- channel assembly
- coiled-coil domain
- subunit interaction
- transient receptor potential melastatin channel (TRPM channel)
The TRPM [TRP (transient receptor potential) melastatin] subfamily comprises a group of eight TRP channel subunits (TRPM1–TRPM8) that display structural relatedness with the putative tumour suppressor melastatin first identified in melanoma cells [1,2]. They are integral membrane proteins and possess a membrane topology similar to that of the well-studied voltage-gated potassium channel subunits; each subunit comprises six membrane-spanning segments with a pore-forming loop region between the fifth and sixth segments, and intracellular N- and C-termini (Figure 1). The N-terminus contains four regions that show sequence similarity but have no defined functions. The C-terminus exhibits common and subunit-specific structural features. While all TRPM subunits contain a TRP motif and a coiled-coil domain, some have unique enzymatic domains in the distal tail, for example, a NUDT9-protein-homologous domain with ADPR (adenosine diphosphoribose) hydrolysis activity in the TRPM2 subunit , or an α-protein kinase domain in the TRPM6 and TRPM7 subunits [5,6]. TRPM subunits form cationic channels (Ca2+-permeable except TRPM4 and TRPM5) that are either constitutively active or activated by numerous physiochemical stimuli and are important in a variety of physiological functions in both excitable and non-excitable cells [1,2].
Functional TRPM channels are analogous to the voltage-gated potassium channels  and TRPV (TRP vanilloid) channels [7–9] in that they are thought to form tetramers through the assembly of four subunits around a central ion-conducting pore. Heterologous expression studies have demonstrated that all the TRPM subunits can form functional homomeric channels (see e.g. [4–6,10–20]). In contrast, TRPM subunit heteromerization is far less clear except for TRPM6 and TRPM7 subunits. Co-expression of the TRPM6 and TRPM7 subunits results in a heteromeric TRPM6–TRPM7 channel that exhibits several functional properties distinct from the homomeric TRPM6 and TRPM7 channels (including sensitivity to pH, modulation by 2-aminoethoxydiphenyl borate and single-channel conductance) .
Subunit interaction is the prerequisite for assembly of multi-subunit channels such as the TRPM channels. Coiled-coil domain is a common protein–protein interaction domain with a helical arrangement comprising heptad (abcdefg) repeats, where positions a and d are preferably occupied by hydrophobic amino acid residues and form the helical interface [21,22]. Previous studies have identified coiled-coil domains and shown that they are essential to direct subunit interaction and tetramerization of several ion channels including the ether-a-go-go K+ channels , M-type KCNQ K+ channels  and TRPV1 channels . As shown in Figure 1, TRPM channel subunits contain a highly conserved C-terminal coiled-coil domain. For the TRPM2 channel subunit, deletion of the coiled-coil domain or replacement with glutamine of the hydrophobic residues in positions a and d of the heptad repeat in the coiled-coil domain severely impairs subunit interaction. Such molecular manipulations also significantly reduce formation of functional TRPM2 channels. The correlated effects on subunit interaction and TRPM2 channel formation suggest that formation of the TRPM2 channel requires subunit interaction mediated by the coiled-coil domain. Moreover, deletion of the coiled-coil domain abolishes the dominant-negative phenotypic functional inhibition of a pore mutation . In a very recent study on TRPM8 channel, Minor and co-workers  show that the coiled-coil domain is indispensable for TRPM8 channel function and formation. Specifically, membrane-tethered TRPM8 coiled-coil domain functions as a dominant-negative inhibitor of TRPM8 channel function. Destabilization of coiled-coil formation by mutating the hydrophobic residues at positions a and d of the heptad repeat reduces TRPM8 subunit oligomerization and channel function. Furthermore, the coiled-coil domains from several TRPM channel subunits (TRPM2, TRPM3 and TRPM6–TRPM8, but not TRPM1), when expressed as fusion proteins, exhibit a strong ability to form tetramers. These studies provide compelling evidence that supports the notion that this highly conserved C-terminal coiled-coil domain represents the key molecular determinant in mediating subunit interaction that is necessary for tetrameric assembly of the TRPM channels. In good accordance with this notion, truncation of most of the TRPM4 C-terminus including the coiled-coil domain, but not deletion of the first conserved N-terminal domain, results in complete loss of the TRPM4 subunit interaction and the TRPM4 channel function .
Identification of endogenously expressed, non-functional truncated subunits or naturally occurring mutant TRPM subunits, and their interaction with and inhibition or disruption of function of the full-length wild-type TRPM channels have revealed an unconventional post-translational regulatory mechanism in which subunit interaction is harnessed to down-regulate the functional activity of TRPM channels. For example, alternative splicing generates a short TRPM1 subunit devoid of the transmembrane domain and the C-terminus that lacks the ability to form functional channels on its own. However, this short TRPM1 subunit can directly interact with and inhibit the functional activity of the full-length subunit to form functional channels by preventing translocation to the plasma membrane . Similarly, an alternative splice variant TRPM2 subunit, which only comprises the N-terminus and the first two transmembrane segments, does not form functional channels on its own, but it can interact with and inhibit the functional activity of the full-length channels by assembling non-functional ‘heteromeric’ channels composed of short and full-length subunits . In addition, single mutation (S141L) in TRPM6 subunit disrupts the TRPM6–TRPM7 heteromeric complex formation, which may underlie the diseased phenotype of hypomagnesaemia with secondary hypocalcaemia . These studies have provided clues to which parts of the TRPM subunits are engaged in subunit interaction and, more importantly, have revealed that subunit interaction can serve as an important mechanism, by which the functional TRPM channel activity is regulated. Although evidence is still lacking, it remains possible that while the C-terminal coiled-coil domain acts as the key molecular determinant for TRPM tetramerization, subunit interactions at other parts such as the N-terminal and/or transmembrane regions may be engaged at a different stage of the TRPM channel assembly, as shown in the voltage-gated potassium channels .
In summary, the past few years have witnessed significant progress in revealing the domains or regions that mediate TRPM subunit interaction and the importance of such subunit interactions in assembly and/or functional regulation of the TRPM channels. However, more studies are needed in order to obtain a comprehensive understanding of the TRPM subunit interaction in the TRPM channel assembly and functional regulation. Many questions remain to be answered. For instance, what is the molecular basis ruling the specificity of subunit interactions governing tetramerization of homomeric and heteromeric TRPM channels? Are additional domains involved in the TRPM8 channel assembly, or which parts are responsible for interaction and formation of the TRPM2 channel in which the coiled-coil domain is absent? What is the mechanism generating short TRPM alternative splice isoforms? Does generation of short alternative splicing subunits operate as a general functional regulatory mechanism among all the TRPM channels?
This work was supported by the Wellcome Trust and the Royal Society. I thank my colleagues, Z.-Z. Mei, H.-J. Mao and X. Rong, for their contributions to the work described here, Professor D.J. Beech for his kind support and Dr C.J. Milligan for her critical comments.
Cell and Molecular Biology of TRP Channels: Biochemical Society Focused Meeting held at University of Bath, U.K., 7–8 September 2006. Organized and Edited by D. Beech (Leeds, U.K.), B. Reaves (Bath, U.K.) and A. Wolstenholme (Bath, U.K.).
Abbreviations: TRP, transient receptor potential; TRPM, TRP melastatin; TRPV, TRP vanilloid
- © 2007 The Biochemical Society