In the heart, Ca entry through Cav channels initiates excitation-contraction coupling, and their dysregulation is important in heart failure. Selective Ca entry is crucial for function of Ca channels because extracellular Na is present at 70-fold higher concentration, yet the structural basis for Ca selectivity is unknown. Building on our determination of the high-resolution structure of a common ancestor of Nav and Cav channels, NavAb, we have now determined the three-dimensional structure of a Ca-selective pore for the first time by constructing a Ca-specific ion selectivity filter in NavAb. This experimental approach will allow high-resolution analysis of structural determinants of Ca binding, selectivity, permeation, and block. Mammalian Cav channels are blocked by Ca antagonist drugs used in therapy of hypertension, angina pectoris, and cardiac arrhythmia. Phenylalkylamines, benzothiazepines, and dihydropyridines bind at three well-characterized receptor sites. Remarkably, Ca antagonist drugs also block CavAb, which therefore provides a structural template for understanding block of Ca channels by drugs that are widely used in treatment of cardiovascular disease. To define the structural basis for pore function and pharmacology of Cav channels, we will address three Specific Aims. 1. We will determine the structural basis for Ca binding and selectivity in CavAb. We will measure the permeability ratio of Ca/Na for NavAb, CavAb, and intermediate mutants. We will determine the structures of the NavAb/CavAb series of mutants in the absence and presence of Ca, identify the binding sites for Ca in the pore, and estimate the relative affinity of Ca for sites in NavAb and CavAb. The affinity for specific binding sites in the pore will be correlated with affinity values estimated from electrophysiological studies and fitting ion conductance measurements to a biophysical model of Ca binding, selectivity, and permeation. 2. We will determine the structural basis for cation block of the CavAb pore. Large divalent and trivalent cations block mammalian Cav channels and CavAb with high affinity. We will measure the affinities of these cations for block of Ca permeation, identify their binding sites in the pore, and compare affinity for binding to specific sites identified by x-ray crystallography with block of Ca permeation. 3. We will explore the structural basis for block of CavAb by Ca antagonist drugs. We will determine the structure of CavAb with Ca antagonist drugs bound in order to understand the molecular basis for channel inhibition at high resolution. We will examine drug binding in CavAb crystals in the presence and absence of Ca in order to understand the complex interactions between Ca binding and drug block. We will create humanized chimeras of CavAb to define the structural basis for high- affinity binding and block of human Cav channels. We will correlate observations of Ca and drug binding in our crystal structures with electrophysiological analysis of drug block of Ca conductance, and we will test biophysical models by fitting kinetics and equilibrium binding parameters to electrophysiological data. Our results will give the first high-resolution insights into Cav channel structure, function, and pharmacology.

Public Health Relevance

Calcium channels are essential for coupling electrical excitation to contraction of the heart, and they are the molecular targets for calcium antagonist drugs used in therapy of hypertension, angina pectoris, and cardiac arrhythmia. This project will give the first atomic-level structural information on the mechanism calcium transport of calcium channels and on the receptor sites and mechanism of action calcium antagonist drugs. This research will be essential for understanding the function of the heart and for structure-based design of new generations of calcium antagonist drugs with improved efficacy and safety.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL117896-01A1
Application #
8629461
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Krull, Holly
Project Start
2014-01-01
Project End
2017-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
1
Fiscal Year
2014
Total Cost
$347,625
Indirect Cost
$122,625
Name
University of Washington
Department
Pharmacology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Tang, Lin; Gamal El-Din, Tamer M; Payandeh, Jian et al. (2014) Structural basis for Ca2+ selectivity of a voltage-gated calcium channel. Nature 505:56-61