The members of the TRP superfamily of cation channels play diverse physiological roles, including receptor- and store-operated Ca2+
entry, mineral absorption, cell death and particularly as sensors for pain, heat, cold, sound, stretch, osmotic changes, etc. The wide variety of physiological functions, in which TRP channels are implicated, underscores diversity in structural and regulatory features. Despite heavily conserved transmembrane domains, the cytoplasmic domains of TRP channels are notably different, which are the target of different gating and regulatory mechanisms (2
). Functional diversity by TRP family members also lies in the ability to hetero-oligomerize, thus creating a wider spectrum of TRP channel phenotypes. Although expected to be tetrameric structures, structural–functional evidence is only beginning to emerge (31
). Moreover, the contribution of each monomeric unit to the functional channel complex is still largely unknown. In the present report, we demonstrated that PC2, TRPC1 and PC2/TRPC1 channel complexes are distinct units consistent with structural tetramers, which functionally correlate with the presence of subconductance states. This is in agreement with our original finding indicating that PC2 contains intrinsic subconductance states, whose resident times, can be modulated by changes in either cytoplasmic pH or holding potential (26
). The presence of subconductance states has also been unmasked by functional inhibition with blocking agents such as amiloride, La3+
and anti-PC2 antibodies (5
). The question we posed in the present study was as to whether each one of the four subconductance states in PC2 has a structural correlation in its monomeric components.
The topology of PC2 and TRPC1 channels was assessed by AFM imaging of lipid bilayer imbedded complexes in solution, thus, providing the first direct comparison between structural and functional correlates of each channel complex. Although many channels are structural tetramers, the evidence for the putative tetrameric conformation of PC2 was heretofore lacking. The correlation between the AFM imaging and the single-channel currents indicates that each isolated monomer usually does not behave as a small conductance channel, but rather that at least four monomers of either protein (PC2 or TRPC1) are required to build a fully functional channel. The time residence and overall conductance at each substate are, therefore, a reflection of the number of contributing units to this tetrameric channel complex. The lack of evidence for isolated ‘monomeric’ (small) channel conductances of PC2 in our preparations supports this contention. Full or partial PC2 channel inhibition by protonation at different pH suggests that in its fully closed state, the four channel subunits are protonated (26
), which can then become sequentially functional, as each one changes conformation. This particular change in conformation is likely to be hindered in the presence of one or more TRPC1 subunits in the complex.
Distinct oligomerization, or ‘aggregation’ domains (AD) (14
) have been identified in the TRP superfamily members, including TRPC, TRPV, TRPM (32
) and even TRPP (PC2) (24
). Both the carboxy- and the amino-termini domains of TRP channels have been implicated in these interactions (34
). These include the so-called TRP domain in the carboxy terminus (32
), and the ankyrin domains found in the amino terminus region of several TRP channels. It is interesting to note, however, that neither one of these domains is present in PC2. Original work by Tsiokas et al
), demonstrated that structural interactions between PC2 and TRPC1 also emanate from the transmembrane domains in PC2, which might be implicated in the aggregation of the hetero-complex. These putative association domains may also have functional correlates. Our findings contend that specific aggregation regions in the monomers of the channel, once oligomerized into a functional complex, can be regulated by protonation, which plays both structural and functional roles by modifying the overall topological and conductive properties of the complex. Protonation in PC2, which we previously identified within the pore and is only reachable from the cytosolic side of the channel (26
), likely helps screen charges and in turn allows conformational changes in the pore structure of the functional complex.
The tetrameric topology of PC2 and TRPC1 found in the current study is consistent with other channels, including the αE
subunit of the Ca2+
) and other TRP channels (13
). However, stoichiometries other than tetramer were also observed for PC2 with both AFM imaging and Western blot analysis. Similar findings have been reported in TRPV6 (15
). FRET analysis of TRP channels further suggests a loosely packed tetramer (13
), which may depend on the aggregation structure of the channel complex. Thus, although our data support a tetramer as the most plausible PC2 functional topology, they do not exclude other topological structures. In light of the present findings, the expectation is that the non-tetrameric TRP channel with either fewer or more interacting subunits, would display, accordingly, different channel conductance and subconductance states. The ubiquitous distribution of TRP channels, including PC2 and TRPC1, and their ability to interact with each other (21
) may underlie their relevance and functional diversity in cell signaling. Our evidence indicates that the presence of at least one, most likely two TRPC1 monomers in the PC2/TRPC1 channel complex, elicits dramatic changes in the single-channel conductance, open probability and responses to pH and amiloride. This, in itself, indicates that the stoichiometric contribution of a particular channel has to be ascertained before any conclusions can be drawn, when assessing TRP-mediated whole-cell currents, or heterologous systems where more than one particular TRP channel is expected.
Our results are in agreement with recent literature, indicating that the isolated proteins have a somewhat similar behavior to cell expression systems. The single-channel conductance of the tetrameric TRPC1 yielded a conductance of 22.8 pS, and a monomeric contribution of approximately 5 pS. This is slightly larger, but in agreement with recently reported data by Du et al
), who showed that TRPC1 in glomerular mesangial cells has a single-channel conductance of 17.2 pS. Bai et al
) reported a TRPC1 conductance of 16 pS. It is important to note, however, that these were patch clamping studies in the biological membrane, where the channel complex may be affected by the associated proteins or structural lipids. Considering the technical and experimental differences, however, it can be accepted as in close agreement. Our data indicate that the interaction between TRPC1 and PC2 in a channel complex modifies the functional properties expected for each one alone. The single-channel conductance of the PC2/TRPC1 hetero-complexes was lower than PC2 homomers but higher than TRPC1 homomers, which is also in agreement with the results obtained by Bai et al
). Moreover, Bai et al
. showed that TRPC1 was significantly activated by addition of the M1 muscarinic agonist oxotremorine-M (Oxo-M) in mIMCD3 cells, but was almost unresponsive to amiloride, a finding similar to ours for the isolated TRPC1 channel. They also demonstrated that co-transfection of PC2 with TRPC1 resulted in the formation of a channel with higher Ca2+
permeability, comparing to either channels alone, which confirms that TRPC1 and PC2 functionally interact to form an heteromultimer, resulting in the modulation of TRPC1 and PC2 properties. Our results, together with the published data, show the dramatic properties changes by heteromultimerization between PC2 and TRPC1.
AFM imaging of TRP (17
) and other channels (37
) has become recently available, which in combination with other high-resolution techniques such as cryo-electron microscopy (40
) has rendered nanometer resolution of a number of channels. For example, Sato's group recently provided evidence for a bell-shaped structure of TRPM2 (40
) and TRPC3 (41
); Barrera et al
. have also observed the tetrameric structure of TRPC1 (17
) and trimeric structure of P2X2 (39
) using AFM imaging. In those studies, however, as in others from the same group (37
), the measurements, which are in strong agreement with their expected theoretical molecular volumes, are mostly consistent with a minimal volume occupied by the particles in solution. Because of the importance of membrane–channel interaction in determining the channel function, we studied the PC2/TRPC1 channel topology in lipid bilayers. The calculated molecular volumes of the PC2 or TRPC1 homomers in our study suggest much larger structures than their theoretical volumes, most likely the result of the conformational changes expected in the transmembrane structure of the channel protein in a lipid bilayer. When compared with more recent information on TRPC3 volume obtained from a molecular reconstruction of frozen hydrated samples by cryoelectron microscopy, for example, the value for TRPC3 is actually 10 times larger than the expected value from its molecular size. Using AFM, syncollin can be observed as a monomer of molecular volume similar to its theoretical value as a naked protein on mica (42
). After insertion of the protein into lipid layers, syncollin shows a doughnut shape consistent with a hexamer. The molecular volume of these structures is, on average, 706 nm3
, which would render a monomer of 118 nm3
, which is almost four times larger than the expected monomer (42
). Recent AFM studies by Shahin et al
) showed that the C2A and C2B domains of synaptotagmin, an integral membrane protein of the synaptic vesicle, dramatically changed in size when imbedded in lipid bilayers. They showed that C2AB has a calculated molecular volume of 80 nm3
, which is in agreement with the AFM measurement of 103 nm3
under ‘naked’ conditions. In lipid bilayers, however, much larger complexes of C2AB were observed. This report, which entails oligomerization (43
), also shows that no particles in the 80 nm3
range are observed, suggesting that even the monomers are likely larger in the membrane. Thus, the calculated molecular volume of channels in lipids is generally larger than the expected theoretical value without lipids. Conversely, it is also possible that the channel insertion to the membrane also alters the lipid topological features and renders extrusions of membrane. Thus, the imaged channel structure in our AFM studies could include topological features from both channel proteins, and surrounding lipid bilayers. Our calculated channel diameters were much larger than those derived from the solution without lipids. Therefore, it is important to begin assessing the contribution of the natural environment, i.e. the lipid bilayer, to the conformational topology of transmembrane proteins such as ion channels. This information is extremely relevant but has only recently begun to emerge. Previous studies from our laboratory (44
) indicated that membrane-imbedded CFTR has a molecular volume larger than its theoretical one. Interestingly, the calculated volume of PC2 is much larger than that of the homomeric TRPC1. This suggests a larger intramolecular space in PC2 when compared with TRPC1, which may help explain the larger pore conductance for PC2 (45
) with respect to TRPC1. This phenomenon, which affects the electrophysiological and regulatory properties of the PC2/TRPC1 heteromeric complex, may also be reflected in the topological structure of the hetero-complex.
In summary, the data in this report indicate that both wild-type PC2 and TRPC1 form homotetramers, with distinct functional and regulatory properties, among which there are the presence of four subconductance states in PC2. The AFM imaging of either of the respective homomers or the PC2/TRPC1 (2:2) hetero-complex is also consistent with a structural tetramer. This structural interaction is reflected in a functional correlation, namely the PC2/TRPC1 heteromers have functional properties, which are distinct from either homomeric structure alone. The AFM imaging also makes evident that the lipid bilayer plays an important role in the topological features of these channel proteins, and supports the contention that structural features in solution of transmembrane proteins are suitably explored in their natural environment. Functional diversity among TRP channels, and in particular the channel properties of PC2 and TRPC1 indeed depend on their mutual interactions, which affect both channels. This type of structural–functional correlation of heterologous oligomerization may be the basis of functional diversity among the TRP family members, and extend to other members, and thus other functions associated with these cellular sensors.