Cell line, system and method for optical control of secondary messengers

Abstract

A variety of methods, devices and compositions are implemented for light-activated molecules. One such method is implemented for generating secondary messengers in a cell. A nucleotide sequence for expressing a chimeric light responsive membrane protein (e.g., rhodopsin) is modified with one or more heterologous receptor subunits {e.g., an adrenergic receptor (alpha1, Beta2)}. The light responsive membrane protein is expresses in a cell for producing a secondary messenger in response to light.

1 .- 12 . (canceled) 
     
     
         13 . A method for generating secondary messengers in a cell, the method comprising:
 a) expressing in the cell a chimeric light-responsive fusion protein comprising a light-responsive rhodopsin-based membrane protein and a heterologous beta2 adrenergic receptor, wherein said expression provides for production of a secondary messenger in response to light, and wherein the chimeric light-responsive fusion protein comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:2;   wherein the cell expresses a secondary messenger-targeted cation channel that is responsive to the secondary messenger; and   b) stimulating the chimeric light-responsive fusion protein with light, thereby generating the secondary messenger in the cell.   
     
     
         14 . The method of  claim 13 , wherein the secondary messenger is cyclic adenosine monophosphate (cAMP). 
     
     
         15 . The method of  claim 13 , wherein said expressing and said stimulating are carried out in vivo. 
     
     
         16 . The method of  claim 13 , wherein said expressing and said stimulating are carried out in vitro. 
     
     
         17 . The method of  claim 13 , wherein the chimeric light-responsive fusion protein is encoded by a nucleotide sequence that is operably linked to a cell type-specific promoter. 
     
     
         18 . The method of  claim 17 , wherein the cell type-specific promoter is a neuron-specific promoter. 
     
     
         19 . The method of  claim 17 , wherein the promoter is a synapsin-1 promoter. 
     
     
         20 . The method of  claim 13 , wherein the cell is a mammalian cell. 
     
     
         21 . The method of  claim 13 , wherein the cell is a neuron. 
     
     
         22 . The method of  claim 13 , wherein the step of stimulating the expressed light responsive membrane protein comprises applying light through an optical fiber. 
     
     
         23 . The method of  claim 13 , wherein the step of stimulating the expressed light responsive membrane protein comprises applying a pulsatile optical stimulus. 
     
     
         24 . A method for assessing the efficacy of a putative treatment regimen relating to intracellular messengers, the method comprising:
 (a) expressing a chimeric light-responsive fusion protein comprising a light-responsive rhodopsin-based membrane protein and a heterologous beta2 adrenergic receptor in a mammalian cell, wherein said expression provides for production of a secondary messenger in response to light, and wherein the chimeric light-responsive fusion protein comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:2;   wherein the cell expresses a secondary messenger-targeted cation channel that is responsive to the secondary messenger;   (b) exposing the chimeric light-responsive fusion protein to light; and   (c) assessing the effects of the treatment by visualizing resultant activity using a cation-sensitive dye.   
     
     
         25 . The method of  claim 24 , wherein the secondary messenger is cyclic adenosine monophosphate (cAMP). 
     
     
         26 . The method of  claim 24 , wherein the chimeric light-responsive fusion protein is encoded by a nucleotide sequence that is operably linked to a cell type-specific promoter. 
     
     
         27 . The method of  claim 26 , wherein the cell type-specific promoter is a neuron-specific promoter. 
     
     
         28 . The method of  claim 26 , wherein the promoter is a synapsin-1 promoter. 
     
     
         29 . The method of  claim 24 , wherein the cell is a neuron. 
     
     
         30 . A chimeric light-responsive fusion protein comprising a light-responsive rhodopsin-based membrane protein and a heterologous beta2 adrenergic receptor, wherein said expression provides for production of a secondary messenger in response to light, and wherein the chimeric light-responsive fusion protein comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:2. 
     
     
         31 . The chimeric light-responsive fusion protein of  claim 30 , wherein expression of the fusion protein in a mammalian cell provides for production of a secondary messenger in response to light. 
     
     
         32 . The chimeric light-responsive fusion protein of  claim 31 , wherein said secondary messenger is cyclic adenosine monophosphate (cAMP). 
     
     
         33 . A nucleic acid comprising a nucleotide sequence encoding the chimeric light-responsive fusion protein of  claim 30 . 
     
     
         34 . The nucleic acid of  claim 33 , wherein the nucleotide sequence is operably linked to a cell type-specific promoter. 
     
     
         35 . The nucleic acid of  claim 34 , wherein the cell type-specific promoter is a neuron-specific promoter. 
     
     
         36 . The nucleic acid of  claim 35 , wherein the promoter is a synapsin-1 promoter. 
     
     
         37 . A recombinant expression vector comprising the nucleic acid of  claim 33 . 
     
     
         38 . A cell genetically modified with the nucleic acid of  claim 33 . 
     
     
         39 . The cell of  claim 38 , wherein the cell is a mammalian cell. 
     
     
         40 . The cell of  claim 38 , wherein the cell is a neuron. 
     
     
         41 . A cell genetically modified with the recombinant expression vector of  claim 37 . 
     
     
         42 . The cell of  claim 41 , wherein the cell is a mammalian cell. 
     
     
         43 . The cell of  claim 38 , wherein the cell is a neuron.