The overall goal of this project is to determine the structural basis for function of Hyperpolarization-activated, Cyclic Nucleotide-gated (HCN) channels. These cation-selective channels conduct the pacemaker current of cardiac myocytes and neurons, and are most closely related to the erg family of K+ channels (e.g., HERG). Together, HCN and HERG channels are important regulators of automaticity in atrial pacemaker cells. However, while HERG (and all other voltage-gated K+ channels) are opened by depolarizataion, HCN channels are opened by hyperpolarization. The molecular mechanism for this difference is unknown. We hypothesize that specific electrostatic and H-bonding interactions between the S4 and other transmembrane domains of HCN facilitate channel opening in response to hyperpolarization, but prevent channel opening in response to depolarization. We further hypothesize that the S4-S5 linker couples the movement of the S4 domains to the activation gate. This hypothesis is based on our discovery that a point mutation (D540K) in the S4-S5 linker of HERG destabilizes the closed state and, similar to HCN, permits channels to open in response to hyperpolarizaiton. Wild-type and mutant HCN and HERG channels will be heterologously expressed in Xenopus oocytes and studied using voltage clamp, substituted cysteine accessibility mutagenesis and biochemical techniques.
The specific aims are to determine the structural basis for hyperpolarization-dependent opening of HCN and mutant HERG channels, to characterize movement of the S4 domain of HCN2 in response to membrane depolarization and hyperpolarization, and to determine the role of electrostatic interactions between S4 and other transmembrane domains in the folding and gating of HCN channels. These studies will elucidate the molecular mechanisms for gating of HCN channels and further our understanding of the cardiac pacemaker.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL065299-03
Application #
6527517
Study Section
Cardiovascular and Renal Study Section (CVB)
Program Officer
Lathrop, David A
Project Start
2000-09-30
Project End
2004-07-31
Budget Start
2002-08-01
Budget End
2003-07-31
Support Year
3
Fiscal Year
2002
Total Cost
$300,000
Indirect Cost
Name
University of Utah
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Abbruzzese, Jennifer; Sachse, Frank B; Tristani-Firouzi, Martin et al. (2010) Modification of hERG1 channel gating by Cd2+. J Gen Physiol 136:203-24
Du, Yi-mei; Zhang, Xiao-xian; Tu, Dan-na et al. (2010) Molecular determinants of Kv1.5 channel block by diphenyl phosphine oxide-1. J Mol Cell Cardiol 48:1111-20
Cheng, Lan; Sanguinetti, Michael C (2009) Niflumic acid alters gating of HCN2 pacemaker channels by interaction with the outer region of S4 voltage sensing domains. Mol Pharmacol 75:1210-21
Brown, Scott; Sonntag, David P; Sanguinetti, Michael C (2008) A highly conserved alanine in the S6 domain of the hERG1 K+ channel is required for normal gating. Cell Physiol Biochem 22:601-10
Decher, Niels; Gonzalez, Teresa; Streit, Anne Kathrin et al. (2008) Structural determinants of Kvbeta1.3-induced channel inactivation: a hairpin modulated by PIP2. EMBO J 27:3164-74
Restier, Lioara; Cheng, Lan; Sanguinetti, Michael C (2008) Mechanisms by which atrial fibrillation-associated mutations in the S1 domain of KCNQ1 slow deactivation of IKs channels. J Physiol 586:4179-91
Piper, David R; Rupp, Jason; Sachse, Frank B et al. (2008) Cooperative interactions between R531 and acidic residues in the voltage sensing module of hERG1 channels. Cell Physiol Biochem 21:37-46
Rosati, Barbara; Dong, Min; Cheng, Lan et al. (2008) Evolution of ventricular myocyte electrophysiology. Physiol Genomics 35:262-72
Cheng, Lan; Kinard, Krista; Rajamani, Ramkumar et al. (2007) Molecular mapping of the binding site for a blocker of hyperpolarization-activated, cyclic nucleotide-modulated pacemaker channels. J Pharmacol Exp Ther 322:931-9
Piper, David R; Hinz, William A; Tallurri, Chandra K et al. (2005) Regional specificity of human ether-a'-go-go-related gene channel activation and inactivation gating. J Biol Chem 280:7206-17

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