丁香园AV

The broad theme of research in the lab is to understand how structurally similar proteins can produce different functions. Understanding such "functional divergence" can help us understand how new functions can evolve through gradual modifications of pre-existing stable protein architectures.

1. Regulation of switching in pacemaker channels

Ion channels called "pacemaker" or "HCN" channels in the heart and brain help regulate oscillations of membrane potential, such as the rhythmic impulse driving the heartbeat. Switching of these channels is regulated by a charged voltage-sensitive domain in the membrane, as well as by binding of cyclic nucleotides (cAMP or cGMP) to a specialized cytoplasmic domain. Artificial compounds that mimic cyclic nucleotides might one day be useful for stabilizing rhythms in patients with disorders like heart arrhythmias or epilepsy.

We have found (see Publications, #1, #7, #9) that the the cytoplasmic cAMP-sensing domain in the pacemaker channel can operate with more than one mechanism, either regulating the extent of channel opening or the speeds of opening and closing. We are further investigating which structures in the channel and which cellular factors contribute to each individual regulatory effect (see Publications #3).

We study channel activity in intact cells as well as in isolated cell-free membranes with control of both cyclic nucleotide concentration and membrane potential. Moreover, a soluble fragment of the HCN channel with the cytoplasmic cAMP-sensing domain can be isolated and its three-dimensional structure has been solved by crystallography (see Publications, #11.) This high-resolution structure will provide valuable mechanistic insights into the interpretation of functional data.

2. Normal and inverse agonism in cyclic nucleotide-gated channels

The "CNG" family of ion channels are an excellent voltage-independent model system for ligand-activation utilizing a cyclic nucleotide-sensing domain similar to that of HCN channels. The opening and closing events of a single CNG channel molecule can be directly observed in real time. Our team (Publications, #6, #8, #10, #12) identified regions within the cytoplasmic domain that control not only the magnitude of the ligand-response but its direction (i.e. enhancing or suppressing opening) in response to the agonist. This is a fascinating structural puzzle, since one cytoplasmic domain is capable of promoting both opening and closing of the membrane-embedded gate for ion flux. Current work analyses the mechanistic role of each of these key regions (see Publications #2).

3. Determinants of efficiency in DNA-repair enzymes

Another line of research focuses on photolyase and cryptochrome, a protein family completely different from ion channels. Photolyase, found in bacteria, enables repair of damage caused by ultraviolet light to DNA, thereby avoiding lethal gene mutations. Bacterial viability can be tested after UV or toxin insults (see Publications #4, #5) to assess survival mechanisms. Cryptochromes have conserved many of photolyase's structural features but lack its DNA-repair capability. We are trying to isolate novel forms of cryptochrome and photolyase that could show repair DNA with different levels of efficiency, and then analyse the structural basis of these functional differences.

We are grateful for funding from agencies including Natural Sciences and Engineering Research Council (NSERC), Michael Smith Foundation for Health Research (MSFHR), and American Heart Association (AHA).

Last Updated - December 2015