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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
HCN pacemaker channels generate the “funny” current that is responsible for initiation and regulation of electrical activity in the heart and the nervous system. Activated near resting membrane potentials, the channel conducts a net inward current that maintains action potential. The direct binding of cyclic AMP upon adrenaline release also enhances channel opening, leading to an increased frequency of action potential. The cytoplasmic cAMP-binding domain is coupled to the C-linker found between the binding domain and the transmembrane domain, subsequently triggering the opening of the pore. Despite extensive research, the mechanism that links the binding event to channel facilitation is still unclear. In this thesis, we looked into the different aspects of the missing link. In addition to basic understanding, the significance of learning about HCN channel modulation by cAMP is that there may be therapeutic advantage of controlling heart rate via drug interaction with the cyclic nucleotide binding pocket. Using the isolated C-linker/binding domain, we pinpointed residues in the binding pocket that contribute to strong affinity and ligand specificity by single residue alanine-scanning and isothermal titration calorimetry for measurement of binding affinity. By comparing our binding data to functional measurements of potency in full-length channels containing the same single substitutions, we found that two residues, L633 and I636, reduced potency more severely than affinity when mutated, and proposed that these residues are involved in a post-binding transition event. We also found that two partial agonists, cCMP and cIMP, bound to the canonical site, but failed to fully promote tetrameric gating ring which is found on the inner side of the pore and hypothesized to facilitate its opening. We proposed that the weakened interactions between the partial agonists and the C-helix of the binding domain limit the formation of the gating ring and led to reduced facilitation of opening in the full-length channel. Finally, we elucidated the mechanism behind two disease-associated mutations found in the cytosolic C-linker/binding domain portion of the HCN2 and HCN4 channels to gain a better understanding of how they influence cAMP binding and channel opening, and cause epilepsy and profound bradycardia, respectively.
Hyperpolarization-activated Cyclic Nucleotide-gated, HCN, channels contribute to the membrane potential of excitable cells including pacemaker cells of the heart and neurons in the brain. By binding to the inner side of the HCN channel, cAMP facilitates channel opening, butthe underlying mechanism has been mainly inferred from relating cAMP concentration to the degree of facilitation. Concentration-response relations reflect the tightly coupled process of cAMP binding and channel opening. The strength of binding and how it is linked to channelopening is not known. Furthermore, cAMP facilitation is not equal among the four mammalianHCN isoforms and the extent to which cAMP binding affinity contributes to these differences isnot known.My experiments support the conclusion that cAMP binds to one site of the isolatedtetrameric C-terminus of HCN2 and HCN4 with high affinity and to three sites with low affinityrevealing negative cooperativity. In contrast, only low affinity binding was observed in HCN1with energetics of binding that were similar to those of the low affinity binding to HCN2. CyclicAMP enhanced oligomerization of the HCN2 C-terminus in solution, but had a negligible effecton oligomerization of the HCN1 C-terminus. Oligomerization in solution is thought to reflect the formation of a gating ring in the intact channel that facilitates opening. Together, this suggests that HCN1 functions as though already disinhibited, explaining its easier opening in the absence of cAMP, its smaller facilitation of opening, and lack of negative cooperativity upon cAMP binding. Lysine substitution at residue 488 of HCN2, initially identified in an individual withidiopathic generalized epilepsy, eliminated negative cooperativity and reduced oligomerizationof the isolated C-terminus upon cAMP binding. This likely reflects a decrease in its ability to form a gating ring in the intact channel and explains the reported inhibition of opening by thismutation.The work presented in this thesis demonstrates the value of studying the C-terminus of the HCN channel in isolation to uncover the mechanism by which the HCN C-terminus and cAMP binding control channel opening that would otherwise be hidden by functional experiments.
The voltage-gated potassium channels of the Kv1 (Shaker-type) family are proteins found in many cell types throughout the body, and are critical for regulating membrane excitability. Ion channel proteins are dynamic by nature, and undergo structural reorientations in response to voltage and other external stimuli. In this thesis, I will describe the results of experiments using the voltage clamp fluorimetry technique, which can relate movements within protein domains to associated electrical behaviour. In Kv1.2 channels, fluorescent emissions from a fluorophore attached to the S4 helix faithfully report the movement of gating charge during depolarization. However, a second phase of fluorescence is also observed which is unique to Kv1.2. Using chimaeras where the external linkers were exchanged between Kv1 homologues, we determined that this phase tracks an interaction between the external linkers which slows channel deactivation. When fluorescence was recorded from Kv1.2 in the presence of the Kvβ1.2 subunit possessing a channel blocking N-terminus, fluorescence was unchanged during activation but slowed during deactivation, suggesting that the blocker sterically hinders activation gate closure and prevents the return of the gating charge. While Kv1.2 requires a Kvβ1 subunit to inactivate, the Drosophila homologue Shaker possesses an N-type inactivation domain on its own N-terminus. Shaker can also inactivate through conformational changes in its selectivity filter, so-called C-type inactivation. This process is structurally linked to activation gate opening, and is accelerated by N-terminal block. We have found that the conformations of the activation gate and the selectivity filter are allosterically linked, and that the N-terminus accelerates C-type inactivation by expelling potassium from a selectivity filter binding site known to inhibit its conformational change. Acceleration of C-type inactivation was also implicated as the mechanism by which an inherited genetic mutation near the Kv1.1 activation gate causes episodic ataxia type-1. However, results from experiments using voltage clamp fluorimetry and single channel patch clamp suggest that accelerated current decay observed in those mutants is more likely due to destabilization of the open state of the activation gate. Taken together, the results of this thesis demonstrate how structural variability between channel homologues leads to their broad functional diversity.
Hyperpolarization-activated cyclic nucleotide gated (HCN) channels are structurallysimilar to voltage gated potassium channels and play pivotal roles in cellular pacemaking. Theirphysiological relevance is illustrated by the fact that genetic mutations in HCN channels areassociated with cardiac arrhythmias. In this study, we performed in-depth evolutionary analysesof HCN channels and functionally characterized the biophysical properties of two novel HCNclones (ciHCNa and ciHCNb) from the urochordate, Ciona intestinalis; a species emerging at thepivotal evolutionary period of invertebrate and vertebrate divergence that occurredapproximately 550MYA. We have expanded the list of known HCN sequences by identifyingand annotating 31 novel genes from invertebrates, urochordates, fish, amphibians, birds, andmammals. Our data suggest that the four vertebrate HCN isoforms arose via three duplicationand diversification events from a single ancestral gene following the divergence of urochordates.Functional analyses of the two ciHCN channels further support this evolutionary trajectory,suggesting that the common single HCN ancestor of urochordates and vertebrates had amammalian-like channel phenotype. Lineage-specific duplication and diversification events and550MY of independent evolution has lead to two Ciona HCN channels with distinct biophysicalproperties.The voltage-gated potassium channels, HERG and KCNQ1, play a key role in cardiacrepolarization. Mutations in these delayed rectifier channels are also associated with cardiacarrhythmias, including long QT syndrome and sudden death. Taking advantage of the >200disease mutations in both of these channels, we performed the first quantitative evolutionary andchemical severity analysis of arrhythmia associated mutations (AAMs). Unlike non-synonymouspolymorphisms (nsSNPs), AAMs are preferentially located to the evolutionarily conserved andfunctionally important sites and regions within HERG and KCNQ1. The mutations are alsochemically more severe than changes which occur throughout evolution. In conjunction withprevious studies, our findings suggest that novel disease-associated mutations can be identifiedby surveying the naturally occurring variation that exists among species. Overall, this thesiscontributes to the current knowledge of the interdependent relationships that exist among ionchannel evolution, ion channel function, and human disease.
Oligomerization and N-linked glycosylation are processes thought to be initiated in the ER during translation and act to regulate the trafficking and functional surface expression of many ion channels and G protein-coupled receptors. HCN channels are known to form tetrameric channels from identical subunits as a prerequisite for functional cell surface expression. Different HCN subunits may also co-assemble to form heteromeric channels with unique properties. Using BRET and immunofluorescence analysis, along with electrophysiology, HCN2 and HCN4 were shown to form functional channels with current properties intermediate of those observed when either isoform is expressed. Furthermore, when expressed in equal amounts in CHO cells, HCN2 and HCN4 did not exhibit preference for homo- versus hetero-oligomerization. Many GPCRs are capable of associating as dimers or higher order oligomers. However the functional and physiological relevance of this type of interaction is not uniform for all GPCRs. The ability of both GIP and GLP-1 receptors to form oligomeric complexes was examined using BRET. The resulting saturation curves suggest that GIPR and GLP-1R are capable of forming receptor homomers and heteromers in CHO cells.The effects of N-linked glycosylation on GPCR trafficking and function are diverse and depend on the receptor studied and whether or not this receptor contains one or more consensus sites for N-glycan binding. Like many family B GPCRs, both the GIP and GLP-1 receptors possess large extracellular N-terminal domains with multiple consensus sites for N-linked glycosylation. Each of these Asn residues was shown to be glycosylated when either human receptor was expressed in CHO cells. Complete removal of N-linked glycosylation severely impaired and completely abolished functional surface expression of GLP-1R and GIPR, respectively. Furthermore, tunicamycin treatment decreased GIPR cell surface number and impaired GIP-potentiated glucose-induced insulin release in an INS-1 pancreatic beta cell line. These results highlight the importance of N-linked glycosylation in regulating the amount of GIPR or GLP-1R at the cell surface. Overall, these results expand upon the diverse roles of oligomerization and N-linked glycosylation in the regulation of membrane protein functional cell surface expression.
To date, the mechanisms and structural determinants which contribute to the regulation of ion channel trafficking, surface expression and function have only been limitedly explored. Through biophysical and molecular characterization of the hyperpolarization-activated cyclic-nucleotide gated channel 2 (HCN2), we have identified a four-amino acid motif (EEYP) in the B-helix of the cyclic-nucleotide binding domain (CNBD) that strongly promotes channel export from the endoplasmic reticulum (ER) and cell surface expression but does not contribute to the inhibition of channel opening. We further demonstrate that this motif augments a step in the trafficking pathway and/or the efficiency of correct folding and assembly. The role of post-translational modifications, specifically N-linked glycosylation, has also been investigated in two different HCN isoforms. All four mammalian HCN channel isoforms have been shown to undergo N-linked glycosylation in the brain. HCN channels have further been suggested to require N-glycosylation for function, a provocative finding that would make them unique in the voltage-gated potassium channel superfamily. Here, we show that both the HCN1 and HCN2 isoforms are also predominantly N-glycosylated in the embryonic heart, where they are found in significant amounts and where HCN-mediated currents are known to regulate beating frequency. Surprisingly, we find that N-glycosylation is not required for HCN2 function, although its cell surface expression is highly dependent on the presence of N-glycans. Comparatively, disruption of N-glycosylation only modestly impacts cell surface expression of HCN1 and leaves permeation and gating functions almost unchanged. The evolutionary significance of this isoforms specific regulation is also examined. Finally, the role of palmitoylation in the regulation of Kv4 channels is examined. Using acylbiotin exchange (ABE) chemistry we are able demonstrate that Kv4.2 is present as a palmitoylated protein in both rat cortical neurons and COS-7 cells. Through mutational analysis of the twelve intracellular cysteine residues within Kv4.2, we were able to localize the site of palmitoylation to the intracellular COOH-terminus. Palmitoylation of Kv4.2 does not contribute to the regulation of activation and inactivation gating parameters. Rather, inhibition of palmitoylation through either mutation of COOH-terminal cysteine residues or the pharmacological agent 2-bromopalmitate results in significant reductions in overall current density measurements.
Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) channels are similar in structure and function to potassium channels. In both, changes in membrane voltage produce directionally similar movement of positively charged residues in the voltage sensor to alter the pore structure at the intracellular side and gate ion flow. Both classes of channels also allow mainly potassium ions to flow, are blocked by cesium ions, and are activated by extracellular potassium. However, HCN channels open when hyperpolarized, whereas most potassium channels open when depolarized. Thus, electromechanical coupling between the voltage sensor and gate is opposite. A key determinant of this coupling is the intrinsic stability of the pore. In potassium channels, the closed, and not the open, pore is more stable, however this it not known for HCN channels. HCN channels are also significantly permeable to sodium despite containing the GYG potassium channel signature selectivity filter sequence. In potassium channels, the selectivity filter sequence is ‘T/S-V/I/L/T-GYG’, which forms a row of four binding sites through which dehydrated potassium ions flow. In HCN channels, the equivalent residues are ‘C-I-GYG’, but whether they form four similarly arrayed cation binding sites is not known. In this thesis, we show using the mammalian HCN2 channel, that the stabilities of the open and closed pore are similar, the voltage sensor must apply force to close the pore, and that the interactions between the pore and voltage-sensor are weak. Furthermore, our data suggest that the conserved cysteine of the selectivity filter does not form a fourth binding site for permeating ions, which prevents it from contributing to either permeation or associated gating functions of the selectivity filter.
Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
The focus of my investigation is the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel, also known as the pacemaker channel. There are four mammalian isoforms (HCN1-HCN4) that share about 60% sequence identity with each other, all activated by hyperpolarization of the membrane potential and permeable to both potassium and sodium. The major differences among four isomers are their responses to binding of cyclic adenosine monophosphate (cAMP), the rate of channel opening and closing, and their dependence on voltage. Recent studies have suggested that the opening and closing of HCN channels involve a step that is voltage independent, which depends upon a region that resides within the S4 and S6 transmembrane region. My study is based upon recent data from the Accili lab in which substitution of a phenylalanine (F) residue near the inner activation gate of the HCN2 channel to alanine (A) dramatically and preferentially slows down channel closing and decreases the dependence of closing on voltage. A 6-state, but not a 4-state, cyclic allosteric model incorporating voltage-dependent transitions moving the channels between resting and active states and voltage-independent transitions between closed and open states was able to describe the complex opening and closing of both the wild type and F/A mutant channels in response to changes in voltage. These models also predicted a significant opening probability between the open and closed states when the channel resides in the resting state.