CellPhys Faculty

Associate Head, Department of Pharmacology & Therapeutics

BSc Hon Class I (Physiology), Leeds University, UK
PhD (Cardiac Electrophysiology), Leeds University, UK
BMBCh (Clinical Medicine), Oxford University, UK

Office: 604-822-5806, Lab: 604-822-3732
Fax: 604-822-6048
Email: fedida@interchange.ubc.ca

Central Theme of the Laboratory

The activity of ion channels and transporters in the heart is central to the normal execution of the inotropic and dromotropic functions of the myocardium. All these proteins are highly regulated and co-assemble with b-subunits or associate with cellular cytoskeletal elements. Our present understanding of the detailed biophysics, the structure-function of these proteins and their cell biology,namely the mechanisms by which they are assembled, processed, and transported to the cell surface are all still poorly understood.

Dysfunction of these proteins, either in inherited diseases or during the pathological processes that underlie arrhythmia and disorders of heart muscle contraction, are a major source of morbidity and mortality in our society today. The central aim of the experimental and modeling work in my laboratory is to develop an in-depth understanding of the ionic mechanisms and underlying cell biology that mediate normal and pathological electrogenesis and mechanical function within the myocardium. The study of cloned cardiac K+ channels in mammalian cell lines provides a powerful model system for detailed studies of the structure and function of human heart voltage-gated K+ channels. These channels underlie the major repolarizing currents in human heart. They affect contractility, the normal propagation of the impulse, the generation of arrhythmias, and their continuation through re-entry. The work in my lab involves study of the human homologues of Shaker Kv1 (Kv1.4 and Kv1.5) and the Shal Kv4 families (Kv4.2 and 4.3), both of which have central roles in human heart action potential (AP) repolarization. The structural differences between the channels leads to functional alterations in their gating properties, and determines their respective roles during the plateau and final repolarization. This also makes them potential targets for different antiarrhythmic drugs. Our approach has been to understand channel structure-function, channel modulation, and drug block by combining mutations of specific amino acid residues within the structural sequence with chemical drug modification.

A major direction has been to understand the mechanisms of inactivation of Kv1 channels, that terminates their activity during the plateau of the AP. We have pioneered measurements of gating currents during human K+ channel activation, and howdrugs interact with channel states intermediate between closed and open. Such information is vital to fully understanding the molecular basis of drug- receptor interactions at sites on ion channels. Along with our experimental methods we have also developed powerful models of channel gating, drug-channel interaction and the mechanisms of action of b-subunits.These models are now at a level of development that they can be used to design further experiments and inform a quantitative approach to our thinking and analysis.

Current Projects

Molecular mechanisms of voltage-gated K (Kv), human ether-go-go (erg) and hyper-polarization-activated channel (HCN) gating. Kv and HCN channels are related in structure but activate in opposite ways. Human ERG (HERG) channels activate in a manner intermediate between Kv and HCN channels. We have made the first measurements of gating currents from human heart Kv channels and are attempting to do the same with HERG and HCN channels, important in controlling action potential duration and pacemaking in heart. We study b-subunit modulation of gating, both of Kv channels and the other two channel types.

Advanced imaging techniques are used to bring rapid time resolution to protein conformational changes during channel gating. Optical methods after labeling of specific channel residues can be used to examine these conformational changes with high time resolution. This enables us to examine both electrically active and electrically silent configurational re-orientations within voltage-gated ion channels.

The experimental problems are addressed using site-directed mutagenesis, gating current measurements and optical techniques. Channel gating in voltage-dependent ion channels is transduced by the movement of fixed charges in the S4 region (of K+ channels) capable of sensing the local potential drop across the lipid bilayer.The study of gating currents resulting from outward movement of these charges and mutational analysis has revealed that a highly conserved region of positively charged residues in the S4 transmembrane region forms the activation voltage sensor of Shaker K+ channels. We have made the first gating current measurements from a human heart K+ channel and developed methodology for over-expression of ion channels to measure such small currents (~1% of ionic current) in human cell expression systems. Gating current measurements, in conjunction with site-directed mutagenesis of putative voltage-sensing domains areused to determine how certain domains and conformational changes of the channel are associated with transfer of charge including those not necessarily associatedwith channel opening. A number of closed-state transitions, influenced by membrane potential can only be seen as gating currents, and some changes in gating, such as those into inactivated states, and recovery, have particular actions on the conformation of returning charge that gives important additional information about the steps involved. Of special interest is to determine the mechanism by which drugs, such as propafenone shift the activation curve to more negative voltages, modulate the channels and bind to different conformational states. Channel and transporter interactions with the cytoskeleton. In collaborative studies already ongoing (Dr Ed Moore / D. Fedida) we have established the first interaction of a cardiac ion channel with the cytoskeleton (FEBS Letters).

We utilize our expertise in fluorescence imaging to determine the localization of channels, and their co-localization with proteins with which they may be functionally coupled. These studies are carried out in myocytes and in cell lines expressing the channel and interacting proteins. Biochemical methods, in combination with electrophysiological and 3-dimensional and confocal immunofluorescence, can be used to examine the co-localization, physical associations and functional consequences of protein-channel interactions in mammalian cells expression systems and in cardiac myocytes. Constructs of channels are expressed in our improved yeast-two-hybrid systemand screened against known cardiac scaffolding elements and against custom designed libraries derived from adult and neonatal cardiac tissues. In addition to two-hybrid assays in yeast, the interactions between cytoskeletal elements and channel/transporter proteins can be investigated in vitro. This is essential to confirm protein-protein interactions directly. The same fragments employed in the two-hybrid assays are expressed in E. coli and tested for their binding abilities in vitro. Regions of the two proteins known already to interact in the two-hybrid system are investigated first. pET vectors are used that also encode "tag" sequences that are useful in protein purification and other procedures. pET21 fuses a T7-tag to proteins expressed from it; pET42 fuses both a GST-tag and a His-tag to proteins expressed from it. All of these tags can be used to facilitate the purifica tion of the proteins once expressed in E. coli. The T7- and GST-tags can also be then be used to immobilize the purified proteins for binding studies. In the picture to the left a C-terminal Kv1.5 channel antibody is used to localize the protein in human embryonic kidney cells.

Imaging Equipment

Our recent success in the Canada Foundation for Innovation competition has allowed us to purchase a confocal microscope to be based at Simon Fraser University (Dr Eric Accili / Dr Glen Tibbits). This will be used for both imaging of fixed tissue, and of live cells using constructs tagged with green fluorescent protein. Funding is also permitting us to complete a fluorescence/electrophysiology apparatus at UBC (Moore/Fedida) that allows combined fluorescence and gating current measurements to be made from ion channels expressed in mammalian cells or oocytes. In addition, funding is provided for additional Silicon Graphics workstations for deconvolution analysis of imaging data (Moore/Tibbits). The Department of Physiology at UBC has given us a dedicated space to set up our fluorescence imaging/ electrophysiology apparatus. This includes an electrically shielded darkroom, so that we can do the electrophysiology within the fluorescence apparatus with only minor shielding around the sensitive equipment. This frees up a lot of space on the rig and improves accessibility. The dedicated space also includes room for new imaging workstations funded by the CFI that we use to streamline analysis of image and current data.

Selected Publications

Kwan DCH, Fedida D & Kehl, SJ, Single channel analysis of the inhibition of hKv1.5 current by external protons. Biophys. J. In press.

Eldstrom J, Yu Q, Martens G, Van Wagoner D, Moore ED, & Fedida, D. Kv1.5 and caveolin-3 do not interact or colocalize in rat or canine cardiac myocytes. J.Physiol. Submitted, 2004.

Fedida D, Zhang S, Kwan D, Eduljee C, & Kehl SJ. Synergistic inhibition of the maximum conductance of Kv1.5 channels by extracellular K+ reduction and acidification. Cell Biochem Biophys. Submitted, 2004.

Zhang S, Eduljee C, Kwan, DCH, Kehl SJ, & Fedida, D. Constitutive inactivation of the hKv1.5 mutant channel, H463G, in K+-free solutions at physiological pH. Cell Biochem. Biophys. 43(2):221-30, 2005.

Brunet S, Aimond F, Li H, Guo W, Eldstrom J, Fedida D, Yamada KA & Nerbonne JM. Heterogeneous expression of repolarizing, voltage-gated K+ currents in adult mouse ventricles. J.Physiol. 559(Pt 1):103-20. Epub June 11, 2004.

Kwan DCH, Eduljee C, Lee L, Zhang S, Fedida, D & Kehl SJ. The external K+ concentration and mutations in the outer pore mouth affect the inhibition of K1.5 current by Ni2+. Biophys. J. 86:2238-2250, 2004.

Kurata HT, Wang Z & Fedida D. Stabilization of inactivation peptide binding by C-type inactivation of Kv channels. J.Gen Physiol. 123, 505-520, 2004.

Lin, S., Wang, Z. & Fedida, D. (2001). Influence of permeating ions on Kv1.5 channel block by nifedipine. Am. J. Physiol. (Heart). 280, H1160-H1172.

Zhang, S., Kwan, D., Fedida, D. & Kehl, S. (2001). External K+ relieves the block, but not the gating shift caused by Zn2+ in human Kv1.5 channels. J. Physiol. 532, 349-358.

Fedida, D. & Hesketh, J.C. (2001). Gating of voltage-dependent potassium channels. Prog. Biophys. Molec Biol. 75, 165-199.

Wang, Z. & Fedida, D. (2001). Gating charge immobilization is caused by the transition between inactivated states in the Kv1.5 channel. Biophys. J. In press, June 2001.

Zhang, S., Kehl, S. & Fedida, D. (2001). Modulation of Kv1.5 potassium channel gating by extracellular zinc. Biophys. J. 81, 125-136.

Cukovic, D., Lu, G, W-K; Wible, B., Steele, D.S. & Fedida, D. (2001). A discrete amino terminal domain of Kv1.5 and Kv1.4 K+ channels interacts with the spectrin repeats of a-actinin-2. FEBS Letters 24928, 1-6.

Kurata, H. T., Soon, G.S. & Fedida, D. (2001). Altered state-dependence of C-type inactivation in the long and short forms of Kv1.5. J. Gen. Physiol. In press, July 2001.