Our long-standing research interests include the regulatory role of the cell’s cytoskeleton in the control of ion channel function.
Horacio F. Cantiello, DVM, PhD
Associate Professor of Medicine
149 13th Street
Charlestown, MA 02129
Our long-standing research interests include the regulatory role of the cell’s cytoskeleton in the control of ion channel function. We have recently focused on the structure-function correlates of disease-associated TRP channels, including polycystin-2 (TRPP2), implicated in autosomal dominant polycystic kidney disease (ADPKD), and its associated channel TRPC1, implicated in mechanosensory functions. We have also studied the channel properties of another disease-associated TRP channel, mucolipin 1 (TRPML1), implicated in the genesis of mucolipidosis type-4 (Research Area A). The cell’s cytoskeleton also plays an important role in the morphogenesis of the primary cilium of epithelial cells, whose dysfunction generates ADPKD as well as other cystic diseases. We have recently explored channel function in the membrane enclosing the primary cilium of renal epithelial cells (Research Area B). Finally, we continue with our studies to address novel properties of cytoskeletal polymers such as actin filaments and microtubules, as potential electrodynamic devices, capable of enabling and processing electrical signals (Research Area C). To accomplish these goals, we apply a number of electrophysiological techniques, including patch clamping and lipid bilayer reconstitution, and novel high resolution imaging techniques, such as atomic force microscopy, which we offer as services to interested parties.
Area (A) of Research. Polycystin-2 (PC2, TRPP2), the gene product of PKD2, whose mutations cause ADPKD, belongs to the superfamily of TRP channels. PC2 behaves as a large conductance, non-selective cation channel, with characteristic subconductance states. We recently explored structural and functional properties of PC2 and whether the conductance substates represent monomeric contributions to the channel complex. To confirm the oligomeric contributions to PC2 channel function, heteromeric PC2/TRPC1 channel complexes were functionally assessed by single channel current analysis.
Amiloride and low pH both inhibited the functional monomeric contribution of PC2, but not TRPC1. Thus, the PC2/TRPC1 hetero-complexes had distinct functional properties different from the homomeric complexes. The topological features of the PC2-, TRPC1- homo-, and PC2/TRPC1 channel complexes, assessed by AFM, and were consistent with structural tetramers (see Fig.). The contribution of individual monomers renders distinct heteromeric channel properties. Our recent studies support tetrameric models of both the PC2 and TRPC1 channels, where the overall conductance of a particular channel will depend on the contribution of the various functional monomers in the complex (from Ref. 4).
Area (B) of Research: The primary cilium of renal epithelial cells is a non-motile sensory organelle, whose role in cell function is associated with mechanosensory transduction signals. We recently demonstrated that renal epithelial primary cilia display abundant channel activity. More recently, we assessed the presence of, and functional signaling pathway associated with, the type-2 vasopressin receptor (V2R) in primary cilia of renal epithelial cells and the mammalian kidney (see Fig.).
We also used confluent LLCPK1 renal epithelial cell monolayers over-expressing a functional V2R-gfp chimera segregated the receptor to both the plasma membrane and the primary cilium. The presence of V2R in primary cilia was confirmed by immunocytochemical analysis of wild type LLC-PK1 cells stained with anti-V2R antibodies and, in LLC-PK1 cells overexpressing the V2R-Flag, with anti-Flag antibody. Ciliary V2R co-localized with adenylyl cyclase type V/VI (AC) in all cells tested. Functional coupling of the receptors was confirmed by measurement of cAMP production in isolated cilia in the presence of vasopressin (10 mM, AVP), and by testing AVP-induced cation-selective channel activity. This is the first demonstration for the presence of vasopressin receptors in primary cilia of renal epithelial cells, and a functional V2R-mediated cAMP signaling pathway, which may help control the sensory function of this organelle.
Area (C) of Research: To further assess the electrodynamic properties of cytoskeletal polymers, bundles of filamentous actin (F-actin) were deposited on a gold-plated surface, which were imaged using AFM and surface potential microscopy (SPM).
The surface potential was mapped as a function of tip distance to surface using a constant bias potential. There was an uneven spatial distribution of charges detected by SPM, consistent with the segmented topological features shown by AFM of the actin bundles. SPM analysis showed localized changes in surface potential between the axial and transversal sections of the bundles, which are consistent with non-uniform charge distributions of adsorbed salt ions on F-actin (from Ref. 2). We also deposited and dried microtubules (MTs) onto a gold-plated surface to image their topology by AFM, and determined their electrical mapping with SPM. We found a strong linear correlation between the magnitude of relative surface potential and MT parameters, including diameter and height (from Ref. 1).
- Zhang P, Cantiello HF. Electrical mapping of microtubular structures by surface potential microscopy. Applied Physics Letters, 9/14/2009, Vol. 95 Issue 11, p113703.
- Zhang P, Cantiello HF. Measurement of charge distribution in actin bundles by surface potential microscopy. Applied Physics Letters, 7/20/2009, Vol. 95 Issue 3, p033701.
- Priel A, Ramos AJ, Tuszynski JA, Cantiello HF. Effect of calcium on electrical energy transfer by microtubules. J Biol Phys. 2008 Oct; 34(5):475-85.
- Zhang P, Luo Y, Chasan B, González-Perrett S, Montalbetti N, Timpanaro GA, Cantero Mdel R, Ramos AJ, Goldmann WH, Zhou J, Cantiello HF. The multimeric structure of polycystin-2 (TRPP2): structural-functional correlates of homo- and hetero-multimers with TRPC1. Hum Mol Genet. 2009 Apr 1; 18(7):1238-51.
- Raychowdhury MK, Ramos AJ, Zhang P, McLaughin M, Dai XQ, Chen XZ, Montalbetti N, Del Rocío Cantero M, Ausiello DA, Cantiello HF. Vasopressin receptor-mediated functional signaling pathway in primary cilia of renal epithelial cells. Am J Physiol Renal Physiol. 2009 Jan; 296(1):F87-97.
- Chen XZ, Li Q, Wu Y, Liang G, Lara CJ, Cantiello HF. Submembraneous microtubule cytoskeleton: interaction of TRPP2 with the cell cytoskeleton. FEBS J. 2008 Oct; 275(19):4675-83.
- Ramos AJ, Cantero MR, Zhang P, Raychowdhury MK, Green A, MacPhee D, Cantiello HF. Morphological and electrical properties of human trophoblast choriocarcinoma, BeWo cells. Placenta. 2008 Jun; 29(6):492-502.
- Montalbetti N, Cantero MR, Dalghi MG, Cantiello HF. Reactive oxygen species inhibit polycystin-2 (TRPP2) cation channel activity in term human syncytiotrophoblast. Placenta. 2008 Jun; 29(6):510-8.
- Dedoussis GV, Luo Y, Starremans P, Rossetti S, Ramos AJ, Cantiello HF, Katsareli E, Ziroyannis P, Lamnissou K, Harris PC, Zhou J. Co-inheritance of a PKD1 mutation and homozygous PKD2 variant: a potential modifier in autosomal dominant polycystic kidney disease. Eur J Clin Invest. 2008 Mar; 38(3):180-90.