METHODS OF IDENTIFYING MODULATORS OF HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATED (HCN) CHANNELS
First Claim
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1. A method for identifying a modulator of a hyperpolarization-activated cyclic nucleotide gated (HCN) channel comprising the steps of:
- (a) providing a cell expressing an HCN channel in a saline solution comprising a membrane potential sensitive dye;
(b) contacting the cell with a test agent;
(c) adding a sodium/potassium diluent to the saline solution of (a) to thereby induce hyperpolarization, wherein the diluent is optionally supplemented with the membrane potential sensitive dye; and
(d) assaying attenuation or enhancement of hyperpolarization, as compared to a control level of hyperpolarization, wherein attenuation of hyperpolarization indicates that the test agent is an HCN channel antagonist, and wherein enhancement of hyperpolarization indicates that the test agent is an HCN channel agonist.
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Abstract
Methods, including high-throughput methods, for identifying modulators of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.
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52 Claims
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1. A method for identifying a modulator of a hyperpolarization-activated cyclic nucleotide gated (HCN) channel comprising the steps of:
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(a) providing a cell expressing an HCN channel in a saline solution comprising a membrane potential sensitive dye; (b) contacting the cell with a test agent; (c) adding a sodium/potassium diluent to the saline solution of (a) to thereby induce hyperpolarization, wherein the diluent is optionally supplemented with the membrane potential sensitive dye; and (d) assaying attenuation or enhancement of hyperpolarization, as compared to a control level of hyperpolarization, wherein attenuation of hyperpolarization indicates that the test agent is an HCN channel antagonist, and wherein enhancement of hyperpolarization indicates that the test agent is an HCN channel agonist. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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46. A method of selecting a cell line that expresses a hyperpolarization-activated cyclic nucleotide gated (HCN) channel comprising the steps of:
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(a) providing a cell suspected to express a hyperpolarization-activated cyclic nucleotide gated (HCN) channel in a saline solution supplemented with a membrane potential sensitive dye (MPSD); (b) adding a sodium/potassium diluent solution to the saline solution of (a) to thereby induce hyperpolarization; and (c) selecting a cell that is hyperpolarized upon addition of the sodium/potassium diluent solution. - View Dependent Claims (47, 48, 49, 50, 51, 52)
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Specification