Christopher Miller is interested in the basic mechanisms by which ion channels and other membrane transport proteins work. These proteins are directly responsible for the ability of nerve cells to generate electrical signals, but are also found in many other biological contexts. A major part of his current research is aimed at the structure and function of anion channels and transporters.
Ion channels are basic elements of molecular hardware in the nervous system. These membrane-spanning proteins directly mediate the transmembrane ionic fluxes giving rise to electrical signals in neurons and other electrically active cells. All proteins of this type have a common structure: that of a water-filled pore spanning the cell membrane. As a result, channels act as "leak" pathways for ions down their transmembrane gradients. The ion channels involved in neuronal function are highly intelligent leaks: they select strongly among the different species of inorganic ions present in the aqueous solutions bathing the cell membrane, and they have the ability to open and close their conduction pores in response to external signals, such as binding of neurotransmitters (ligand-gated channels) or changes in transmembrane electric field (voltage-dependent channels).
My research is aimed at questions of fundamental molecular mechanisms of ion channel operation and the underlying protein structures involved. Until recently, when the first direct structure determinations of ion channels began to emerge, it was necessary to draw structural inferences from close examination of ion channel function. This can be done because ion channels can be studied at the single-molecule level, both in the cellular environment (using patch-recording techniques) or after reconstitution into biochemically defined artificial membranes. These high-resolution recording methods can now be combined with direct structure determination to illuminate the operations of these membrane proteins. My lab is focusing on several membrane proteins that provide opportunities to address basic questions about the structures and mechanisms of ion channels and membrane transporters for ionic substrates.
Cl– Channels and Transporters in the Androgynous CLC Family
A class of Cl–-conducting ion channels has recently been recognized as centrally involved in many cellular electrical processes. This large molecular family of CLC-type Cl– channels is expressed in nearly all cells. For example, CLC channels set the electrical excitability of mammalian skeletal muscle, permit acidification of endosomes, and regulate blood volume (and hence pressure) via renal epithelial transport. Certain inherited human myotonias and several human renal and bone diseases result from disruption of CLC channel genes. Several years ago, we identified CLC homologs in bacterial and archaeal genomes and showed that one of these conducts Cl– when reconstituted in liposomes. The high-resolution structure of this bacterial homolog was determined in Roderick MacKinnon's lab (HHMI, Rockefeller University). We recently stumbled upon a startling conclusion: the bacterial homolog is not an ion channel at all. Instead, it moves Cl– ions across the membrane by an entirely different mechanism: stoichiometric exchange for protons. Thus, the CLC molecular family is androgynous, containing isoforms of two mechanistically different types, Cl– channels and Cl–/H+ exchange-transporters. This situation is now recognized to be a general feature of eukaryotic CLCs as well. The human genome contains nine CLCs: four channels and five transporters. We are currently trying to understand the mechanistic implications of this unusual finding.
We are examining the mechanism by which the bacterial CLC protein couples Cl– to H+ movement, by relating the electrophysiological and kinetic behavior of functionally altered mutants to their x-ray crystal structures. HHMI associate Hyun-Ho Lim is examining water-mediated proton movements through the transporter protein in exchange with Cl–.
Ion Transport in Bacterial Extreme Acid Resistance
The extreme acid resistance (XAR) response by which enteric bacteria such as Escherichia coli protect themselves against the low pH of the stomach involves several amino acid decarboxylase genes working in concert with several membrane transport proteins (including the CLC Cl– transporter). Among these is an arginine transporter of the APC superfamily that functions physiologically as a virtual proton pump to prevent acid overload of the cytoplasm during acid shock. HHMI associates Yiling Fang and Hariharan Jayaram determined the structure of this transporter as a first step toward understanding the mechanism by which the protein selects finely for substrates—an essential requirement for its proton-extrusion function. HHMI associate Ming-Feng Tsai is testing mechanisms of substrate recognition suggested by crystal structures and examining the strange, low-pH physiology of bacteria mounting the XAR response.
Two Families of Novel Membrane Exporters for F –
Our biosphere is bathed in F– ions. Natural levels of F– in groundwater, sea, and soil are typically in the 10- to 100-μM range, and fluoridation of drinking water for dental health in developed countries adds another 50–100 μM. Although F– at these levels in vitro inhibits enzymes essential for glycolysis (enolase) and for DNA and RNA synthesis (pyrophosphatase), the anion is not a threat to vertebrates. However, many bacteria, unicellular eukaryotes (including pathogenic fungi and protozoa), and green plants carry genes for F–-specific membrane exporters that keep cytoplasmic F– low. These exporters, recently discovered by Ronald Breaker's group (HHMI, Yale University), are found in two distinct, unrelated molecular families of integral membrane proteins: a specific CLCF clade of the ubiquitous CLC superfamily, and a family of small membrane proteins, previously unannotated in the database, which we denote "Fluc" proteins. HHMI associate Randy Stockbridge and graduate student Ashley Brammer have screened more than 30 homologs from these families, have established their general ability to protect bacteria from F– toxicity, and have found roughly 10 biochemically tractable homologs that may be purified, reconstituted, and functionally examined. The CLCF proteins are F–/H+ antiporters, while the Fluc proteins are F–-specific ion channels. We are currently beginning detailed mechanistic investigation of these different modes of F– transport across biological membranes.
As of March 23, 2016