Modified cyclic nucleotide gated ION channels
AssigneeUniversity of Colorado Board of Regents
InventorsJeffrey W. Karpen, Thomas C. Rich, Dermot M.F. Cooper, Jerome Schaack
Life of this patent
Estimating the term…
Abstract
The present invention provides modified cyclic nucleotide gated (CNG) channels. In particularly preferred embodiments, the modified CNG channels exhibit increased sensitivity and specificity for cAMP, as compared to wild-type CNG channels. In additional embodiments, regulation by Ca2+-calmodulin has been removed in the modified CNG channels. Convenient optical methods for detecting changes in cAMP, taking advantage of the Ca2+ permeability of the channel are also provided by the present invention. In addition, electrophysiological methods are further provided.
Claims25
An isolated nucleic acid encoding a modified olfactory cyclic nucleotide-gated ion channel, wherein said channel comprises mutations which together impart increased cAMP sensitivity, decreased cGMP sensitivity, decreased nitric oxide sensitivity and decreased calcium-calmodulin sensitivity.
An isolated nucleic acid encoding a modified olfactory cyclic nucleotide-gated ion channel, wherein said channel comprises at least one mutation selected from the group consisting of a C460W mutation and a E583M mutation.
A method for determining local intracellular cAMP concentration within a eukaryotic cell, comprising the steps of:
- a) providing:
- i) at least one eukaryotic cell, and
- ii) a nucleic acid encoding a modified olfactory cyclic nucleotide-gated ion channel, wherein said channel comprises at least one mutation selected from the group consisting of a C460W mutation and a E583M mutation;
- and
- b) contacting said eukaryotic cell with said nucleic acid under conditions suitable for expressing said modified olfactory cyclic nucleotide-gated ion channel in said eukaryotic cell;
- c) measuring intracellular calcium concentration in said eukaryotic cell;
- and
- d) determining local intracellular cAMP concentration based upon said intracellular calcium concentration.
A method for determining local intracellular cAMP concentration within a eukaryotic cell, comprising the steps of:
- a) providing:
- i) at least one eukaryotic cell, and
- ii) a nucleic acid encoding a modified olfactory cyclic nucleotide-gated ion channel, wherein said channel comprises at least one mutation selected from the group consisting of a C460W mutation and a E583M mutation;
- and
- b) contacting said eukaryotic cell with said nucleic acid under conditions suitable for expressing said modified olfactory cyclic nucleotide-gated ion channel in said eukaryotic cell;
- c) measuring the electric current across the plasma membrane of said eukaryotic cell;
- and
- d) determining local intracellular cAMP concentration based upon said electric current.
A method for determining whether a candidate compound is capable of modulating local intracellular cAMP concentration within a eukaryotic cell, comprising the steps of:
- a) providing:
- i) at least one eukaryotic cell expressing a modified olfactory cyclic nucleotide-gated ion channel, wherein said channel comprises at least one mutation selected from the group consisting of a C460W mutation and a E583M mutation, and
- ii) a drug candidate;
- and
- b) determining local intracellular cAMP concentration within said eukaryotic cell in the presence and absence of said drug candidate.
Dependencies are inferred from the claim text.