Structure, function, regulation and cell biology of ion channels.
We study the structure, function, regulation and cell biology of ion channels.
What are ion channels and what do they do?
Ion channels are multi-subunit membrane protein complexes. They selectively conduct ions across cell membranes when activated, resulting in electrical and/or chemical changes in cells. As such, ion channels are essential for all our body functions. Genetic mutations in or malfunction of ion channels cause numerous human diseases and disorders, such as heart diseases, epilepsy, cystic fibrosis, migraine and autism, to name just a few.
We focus on voltage-gated calcium channels (VGCCs) and transient receptor potential (TRP) channels. Both types of channels, when open, lead to membrane depolarization and calcium influx; thus, they not only increase cell excitability but also affect cellular calcium signaling. VGCCs are present in neurons, muscles and other excitable cells, and they open in response to membrane depolarization. They are vital for diverse biological processes including muscle contraction, neurotransmission, neurodevelopment and gene expression. TRP channels are more ubiquitous and are activated by more diverse mechanisms, including intracellular or extracellular ligands, mechanical stretch, temperature as well as changes in membrane voltage. TRP channels are involved in numerous biological processes, and they play an especially important role in vision, taste, hearing, touch, olfaction, temperature sensation, and other senses.
We use a combination of techniques including molecular biology, biochemistry, patch-clamp, x-ray crystallography and optical imaging to study the structure, function, regulation, targeting, localization and activity-dependent trafficking of VGCCs and TRP channels. Our objectives are to better understand how these ion channels work as molecular machines, how they function to control cell excitability and signaling, and how their mutations and malfunction cause human diseases.
Current and Future Research Topics
Cell biology of VGCCs
Different types of VGCCs display distinct albeit overlapping subcellular localization and physiological roles. Thus, L-type channels are localized in the soma and dendrites and regulate neuronal excitability and gene transcription, whereas N- and P/Q-type channels are concentrated at presynaptic nerve terminals and mediate neurotransmitter release. Location governs function. We are therefore very interested in studying the molecular mechanisms of targeting, localization and activity-dependent trafficking of different types of VGCCs. The tools we use include mouse genetics, confocal and two-photon microscopy, biochemistry and electrophysiology.
Regulation of VGCCs
The activity of VGCCs is regulated by numerous signaling pathways and proteins, including phosphorylation/dephosphorylation, membrane lipids, G proteins and calmodulin (CaM). We are studying the molecular and biophysical mechanisms of regulation of P/Q-type VGCCs by G protein βγ subunits (Gβγ), by Gem, a member of the RGK family of Ras-related small GTP-binding proteins, and by Pax6(S), a novel primate-specific isoform of the transcription factor Pax6 that we cloned and found to interact with the beta subunit of VGCCs.
Regulation of TRPC channels
The canonical TRPC channels are widely distributed and have diverse biological functions, including being involved in the fear response. They are activated by the stimulation of phospholipase C-coupled receptors, resulting in membrane depolarization and Ca2+ influx, which in turn feedback to regulate the channel activity, either positively or negatively, through the Ca2+-binding protein CaM. TRPC subunits contain one to four putative CaM-binding sites. Combining x-ray crystallography, biochemistry, yeast-two hybrid screen and patch-clamp, we are studying the structural and molecular basis of CaM regulation of several types of TRPC channels.
Assembly of TRPP/PKD complexes
TRPP channel subunits assemble with PKD (polycystic kidney disease) family of proteins to form functionally important receptor/ion channel complexes. For example, TRPP2 associates directly with PKD1, which is a large (~465 kDa) integral membrane protein with 11 transmembrane regions and is likely a cell surface receptor for extracellular ligands and matrix proteins. Mutations in TRPP2 and PKD1 cause autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases in humans. TRPP3 associates directly with PKD1L3, which belongs to the same family and has the same transmembrane topology as PKD1 does. The TRPP3/PKD1L3 complex forms the sour taste receptor and probably an acid-sensing receptor in the brain. We are using a multipronged approach including biochemistry, single molecule optical imaging and x-ray crystallography to study the structural and molecular basis of the assembly and subunit stoichiometry of the TRPP2/PKD1 and TRPP3/PKD1L3 complexes.
Crystal structure of VGCCs and TRP channels
To fully understand how VGCCs and TRP channels work, are regulated and function physiologically, it is necessary to obtain high-resolution structures of these channels, alone and in complex with their regulatory proteins. We are ultimately interested in the structure of the full length channels, but at present we are concentrating on solving the crystal structure of various intracellular or extracellular domains of VGCCs and TRP channels. These domains often interface with other regulatory or scaffolding proteins and are critical for channel function and regulation. The channels we are working on now include L- and P/Q-type VGCCs and TRPC and TRPP channels.
- Yu, Y., Ulbrich, M.H., Dobbins, S. Li, M-h., Zhang, W.K., Tong, L., Isacoff, E.Y., and Yang, J. (2012) Molecular mechanism of the assembly of an acid-sensing receptor/ion channel complex Nat. Commun. 3: 1252. doi: 10.1038/ncomms2257.
- Fan, M-m., Zhang, W.K., Buraei, Z., and Yang, J. (2012) Molecular determinants of Gem protein inhibition of P/Q-type Ca2+ channels. J. Biol. Chem 287: 22749-22758.
- Li, M-h., Yu, Y., and Yang, J. (2011) Structural biology of TRP channels. Adv. Exp. Med. Biol. 704: 1-23.
- Zhu, J., Yu, Y., Ulbrich, M.H., Li, M-h., Isacoff, E.Y., Honig, B., and Yang, J. (2011) A structural model of the TRPP2/PKD1 C-terminal coiled-coil complex produced by a combined computational and experimental approach. Proc. Natl. Acad. Sci 108: 10133-10138..
- Fan, M-m., Buraei, Z., Luo, H-R., Levenson-Palmer, R., and Yang, J. (2010) Direct inhibition of P/Q-type voltage-gated Ca2+ channels by Gem does not require a direct Gem/Cavβ interaction. Proc. Natl. Acad. Sci 107: 14887-14892.
- Buraei, Z., and Yang, J. (2010) The β subunit of voltage-gated Ca2+ channels. Physiological Reviews 90: 1461-1506.
- Yu, Y., Ulbrich, M.H., Li, M-h., Chen, X-Z., Ong, A.C.M., Tong, L., Isacoff, E.Y., and Yang, J. (2009) Structural and molecular basis of the assembly of the TRPP2/PKD1 complex. Proc. Natl. Acad. Sci 106: 11558-11563.
- Zhang, Y., Chen, Y-h., Bangaru, S.D., Abele, K., Tanabe, S., Kozasa, T., and Yang, J. (2008) Origin of the voltage-dependence of G protein regulation of P/Q-type Ca2+ channels. J. Neurosci 28: 14176-14188.