Interactions of the Novel HCN4-Associated Proteins Hint1 and Hint2 with HCN4 Ion Channels Mallory Myers, B.A.1, Colin Peters, Ph.D.2, Hicham Bichraoui, Ph.D.2, Catherine Proenza, Ph.D.2,3 1University of Colorado School of Medicine, 2Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, 3Department of Cardiology, University of Colorado Anschutz Medical Campus INTRODUCTION HYPOTHESIS RESULTS kDa Heart rate is driven by the sinoatrial (SA) node. SA node cells have ion Hint1 and Hint2 physically interact with HCN4. channels in their membranes that allow them to generate an electrical Anti-HCN4 Anti-GFP impulse that makes the heart contract. One important channel is the kDa 5 µL 10 µL kDa 5 µL 10 µL 20 µL hyperpolarization-activated cyclic nucleotide-gated channel (HCN), METHODS which generates the funny current. The funny current initiates a gradual depolarization that eventually triggers action potential firing. Cell lines A B Fig. 13. Co-IP using anti-HCN4 antibodies. Membrane incubated with Fig. 9. Confirmed expression of HCN4 and GFP-Hint1 in HEK cell anti-Hint2 antibodies. lysates. (A) HCN4 at ~150 kDa. (B) GFP-Hint1 at ~86 kDa. HEK cells + HCN4 GFP-Hint1 Na+ influx DNA kDa (If) HCN4+ HEK cells A expressing GFP-Hint1 Fig. 1. Cardiac conduction system. Fig. 2. Pacemaker action potential. kDa Fig. 5. GFP-tagged Hint1 was transfected into HEK cells expressing HCN4. Of the four HCN isotypes, HCN4 is the most abundant in the SA node and has been implicated in: Atrial fibrillation Heart block Sudden Infant Death Syndrome (SIDS) CHO cells Fig. 14. Lanes 1-6 are probed with anti-HCN4 antibodies. Lanes 7-8 are Brugada syndrome Inappropriate sinus Tachycardia-bradycardia + HCN4 Tetracycline B probed with anti-Hint2 antibodies. tachycardia syndrome Fig. 6. We grew CHO cells stably expressing HCN4 and endogenous Hint2. kDa Familial sinus Sick sinus syndrome Ventricular fibrillation Tetracycline induces HCN4 expression in CHO cells. bradycardia DISCUSSION One important feature of the HCN channel is the cyclic nucleotide binding Confirming expression Does Hint1 physically interact with HCN4? domain (CNBD). In the absence of cyclic adenosine monophosphate Results were mixed. Fig. 10b suggests no, but Fig. 11 suggests yes. (cAMP), the CNBD inhibits channel activation, a phenomenon called Substrate reagent Unfortunately, both results had bands in the control, so we cannot make “autoinhibition.” cAMP, when present, directly binds the CNBD and relieves Fig. 10. Co-IP with anti-HCN4 antibodies. (A) Membrane probed with any conclusions. autoinhibition, allowing the channel to open at more positive voltages. Secondary ab Enzyme anti-HCN4 antibodies. (B) Membrane probed with anti-GFP antibodies. WE = whole extract (HEK HCN4 lysate), FT = flow through, W2 = second Does Hint2 physically interact with HCN4? wash, E1 = first eluate, and E2 = second eluate. Yes. Three separate co-IPs show evidence of physical interaction in CHO HCN4 + Tetracycline cells (Figs. 12-14). Primary ab Gel Membrane Blocking reagent Limitations • Could not use anti-Hint1 antibody due to non-specific binding. • Anti-GFP antibody is non-specific (recognizes many bands). Fig. 3. The HCN channel. Fig. 7. Using SDS-PAGE and Western blots, we confirmed expression of kDa • When Hint1 is tagged with GFP, it no longer affects HCN4 activity in HCN4 and GFP-Hint1 in HEK cells, and expression of HCN4 and Hint2 in patch-clamping studies, so GFP tag may prevent Hint1 from physically When HCN4 is expressed in Chinese hamster ovary (CHO) cells, cAMP HEK and CHO cells. interacting with HCN4. does not change HCN4’s activation, and there is relief of autoinhibition without cAMP. Our lab identified two potential interaction partners – Hint1 Future Directions and Hint2 – which could contribute to this phenomenon. Transiently Co-immunoprecipitations (coIPs) • Tag Hint1 with Myc → perform more co-IPs to look for physical interaction between Hint1 and HCN4. transfecting Hint1 or Hint2 into HCN4-positive HEK cells changes the A B voltage activation curve of HCN4. Hint1 abolishes the positive voltage shift Interacting protein Fig. 11. Co-IP with anti-GFP antibodies. Membrane probed with anti- • Identify specific domains of interaction between Hint2 and HCN4. HEK cell lysate Ab cross-linked to beads bound to ab + bead associated with cAMP, while Hint2 causes a positive voltage shift like HCN4 antibodies. CWE = control whole extract. cAMP. How they interact with HCN4 and exert this effect is unknown. REFERENCES 1. Lilly, L. S. (2016) Pathophysiology of heart disease: a collaborative project of medical students and faculty. 6th A 1.0 B C ed. Wolters Kluwer/Lippincott Williams & Wilkins. Baltimore, MD. 2. Harzheim, D. et al. Cardiac pacemaker function of HCN4 channels in mice is confined to embryonic development and requires cyclic AMP. EMBO J 27, 692-703 (2008). HCN4+cAMP -100 0.8 Bead + Rabbit kDa 3. Milanesi, R. et al. Familial sinus bradycardia associated with a mutation in the cardiac pacemaker channel. N anti-HCN4 C D Engl J Med 354, 151-157 (2006). 4. DiFrancesco, D. Funny channel gene mutations associated with arrhythmias. J Physiol 591, 4117-4124 (2013). 0.6 -105 Antigen (HCN4) 5. Verkerk, A. & Wilders, R. Pacemaker activity of the human sinoatrial node: an update on the effects of mutations Max HCN4+Hint1 in HCN4 on the hyperpolarization-activated current. Int J Mol Sci 16, 3071-3094 (2015). G/G 0.4 Interacting protein +cAMP -110 6. Servatius, H. et al. Phenotypic Spectrum of HCN4 Mutations: A Clinical Case. Circ Genom Precis Med 11, HCN4 (GFP-Hint1) doi:10.1161/CIRCGEN.117.002033 (2018). 0.2 HCN4+cAMP Flow through Eluate 7. DiFrancesco, D. & Tortora, P. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. HCN4+HINT1 -115 (Ab + interacting protein) Nature 351, 145-147 (1991). HCN4+HINT1+cAMP (Non-interacting proteins) 8. Bichraoui, H. et al. A Novel HCN4 Interacting Protein in CHO Cells and Sinoatrial Node Myocytes. Biophysical 0.0 ActivationMidpoint (mV) Society 61st Annual Meeting, New Orleans, LA, February 2017. -160 -140 -120 -100 -80 -60 -120 Fig. 8. (A) Cells were lysed with a lysis buffer. (B) Antibodies were cross- Membrane Potential (mV) linked to beads. Then, the lysed cell mixture was added to the beads. (C) The ACKNOWLEDGEMENTS Fig. 4. (A) Conductance-voltage relationships of HCN4 stably expressed in beads were centrifuged; the supernatant was taken as the flow through. The Proenza Lab HEK cells in the absence or presence of Hint1 and cAMP. (B) HCN4 beads were then washed and centrifuged three times. (D) The beads were Fig. 12. Co-IP with anti-Hint2 antibodies. Hint2 is transfected into HEK • Christian Rickert, Ph.D. – Postdoctoral Fellow currents with cAMP. The red trace is hyperpolarization to -110 mV. (C) heated and then centrifuged. The supernatant was taken as the eluate. cells but endogenous in CHO cells. Membrane probed with anti-HCN4 • Dan Bartos, Ph.D. – Postdoctoral Fellow Average midpoint of activation for HCN4 in the absence or presence of Analysis of the samples was done via SDS-PAGE and Western blots. antibody. H4 = expressing HCN4 and T = tetracycline. Hint1 and cAMP. • Emily Sharpe, Ph.D. – Doctoral Student RESEARCH POSTER PRESENTATION DESIGN © 2019 www.PosterPresentations.com.