Structure and Function of Membrane Proteins Responsible for Intracellular Signaling

Proteins of the cellular membranes provide crucial functions in the body; they are the passageways into and out of the cell, the pumps that generate and maintain chemical and electrical gradients, the receptors that transduce chemical messages and the scaffolding that gives the cell its shape. In order to function, proteins have a very specific shape which forms domains for interaction; the domains of a protein may interact with small molecules and make a structural rearrangement, or they may mediate protein-protein interactions or be assembled with other protein subunits to form a protein complex. Proteins with complicated function often are arranged into protein complexes which provide modular subunits that can be switched in or out of the complex over time or across cells. This modularity allows for combinatorial regulation of the output of the protein. In the case of ion channels--proteins that allow the passage of ions from one side of the membrane to another--the particular number of subunits and the combination with which different types of subunits may be assembled determine biophysical properties, such as the opening cooperativity or the current amplitude of the channel./nIonotropic glutamate receptors are an important class of ion channels that open in response to glutamate. Glutamate receptors are the primary excitatory receptors of the mammalian central nervous system and they receive chemical signals which they convert to an electrical signal. The glutamate receptors are ion channels composed of various glutamate receptor subunits, which determine the functional output. For example, some subunits only allow permeation of certain ions, like sodium or potassium, but others allow for calcium influx. Similarly, some subunits have faster or slower gating kinetics, which determines the current amplitude in response to glutamate. In principle, glutamate receptor subunits may be similar enough that any combinatorial assembly of the subunits would be possible; however, it is known that glutamate receptors have a highly regulated assembly process that limits the possible combinations available to the cell. Using a single-molecule technique called subunit counting, we determined the rules of assembly for a kainite-type glutamate receptor. We discovered that that GluK2 homotetramers and 2:2 GluK2/GluK5 heterotetramers are possible receptor combinations, but not 1:3 or 3:1 GluK2/GluK5 assemblies. This suggests that there are specific regulation steps in the assembly of the quaternary structure that control the number and types of subunits assembled into a finished glutamate receptor./nOnce a functional glutamate receptor has been properly assembled and delivered to the membrane, they are subject to various regulatory processes. One regulatory mechanism relies upon a protein family called the transmembrane AMPA-R regulatory protein (TARP) family. TARPs traffic AMPA-type glutamate receptors to the plasma membrane of cells and can functionally regulate the receptors by slowing receptor deactivation and desensitization. The molecular details of this process were previously unknown, but we have determined that TARPs bind to AMPA receptors in an expression-dependent manner. For low expression densities of the TARP, zero or one TARP will bind to the glutamate receptor. Alternatively, higher expression will yield more TARPs bound, and in the case of stargazin, the canonical member of the TARP family, it can bind up to four to a single glutamate receptor. This suggests that the mechanism of AMPA receptor regulation by TARPs is controlled at the level of plasma membrane expression of the TARPs./nOther ion channels, instead of responding to chemical signals, are activated by the voltage across the membrane. For example, the voltage-gated calcium channel is an important source of calcium entry into the cell and is therefore a key molecule in intracellular signaling. Calcium entry is tightly regulated by processes of calcium-dependent inactivation and calcium-dependent facilitation. These processes are regulated at the C-terminus of the voltage-gated calcium channel where calmodulin, a calcium-binding protein, is bound. To study the structural details of the calcium regulation process, the molecular structure of the voltage-sensitive calcium channel C-terminus was determined by protein crystallography and individual C-termini were seen to associate with a partner C-terminus. These data suggest that, contrary to popular belief, voltage-gated calcium channels may multimerize to form channel dimers, which may have additional calcium entry regulation. However, using subunit counting, we determined that voltage-gated calcium channels are, in fact, protein monomers and overexpression of calmodulin does not mediate dimerization./nMembrane proteins are also capable of signaling between the different membrane compartments of the cell. STIM1, a calcium-sensing protein of the endoplasmic reticulum (ER), signals from the ER membrane to proteins of the plasma membrane. These protein targets include the Orai1 calcium channel, for the restocking of ER calcium, and adenylyl cyclase to stimulate the production of cAMP. Using total internal reflection fluorescence (TIRF) microscopy and electrophysiology, we elucidate the pathway by which the innate immune system of airway epithelial cells responds to a bacterial lactone. Specifically, IP3 receptors become activated which releases calcium from the ER. Next, STIM1 activates adenylyl cyclase which signals to the cystic fibrosis transmembrane conductance regulator (CFTR) via cAMP and PKA. The activation of CFTR produces a transepithelial current to cleanse the epithelial cells of any bacteria that may be present.

Advisor: Isacoff, Ehud Y