Introduction/Overview
Cells are studded with millions of special proteins
that control the passage of ions across the surface membrane. Ion
flow into or out of cells underlies vital functions like electrical
signaling in neurons and in heart and muscle cells, secretion of hormones
and neurotransmitters, cell volume regulation, kidney function, and
fertilization. Two principal classes of membrane proteins regulate
ion movements: ion-motive pumps and ion channels. Though both provide
a favorable environment for shepherding ions across the otherwise
impenetrable phospholipid bilayer, ion pumps and channels are traditionally
viewed as different entities. But our work has led us to believe that
pumps and channels are far more closely related than generally assumed.
Our two subjects are the Na+,K+-ATPase (or Na+/K+ exchange pump) and
the cystic fibrosis Cl- ion channel, CFTR (Cystic Fibrosis Transmembrane
conductance Regulator), both of biomedical importance. The Na+/K+
pump extrudes 3 Na+ from a cell and recovers 2 K+ at the expense of
a single molecule of ATP, so establishing the transmembrane gradients
of Na+ and K+ ions that are crucial to cell life. Mutations in the
gene that encodes CFTR cause cystic fibrosis, the most prevalent lethal
genetic disease in the USA. Our goal is to learn how these two molecular
machines work.
Background
Why are ion pumps and ion channels perceived
as being so different? A major reason is that the ion-selective pore
in a channel conducts dissipative ion flow, down the electrochemical
gradient, at a high rate (e.g. 107 to 108 s-1),
whereas an ATPase pump moves ions against their electrochemical gradient,
and far more slowly (e.g. ~102 s-1). But the high
rates apply only to open ion channels, and all channels contain a gate
that allows the ion flow to be turned on and off as needed. Opening
and closing of the gate involve substantial conformational changes that
are relatively slow (~102 to 103 s-1)
and require input of energy. Energy sources include movement within
the membrane's electrical field of charged residues in voltage-gated
channels, or binding of an extra- or intracellular messenger in ligand-gated
channels, or physical distortion in mechanosensitive channels. Nevertheless,
the conformational changes that accompany opening and closing of an
ion channel might not differ greatly from those associated with the
ion movements effected by a pump. In fact, we suggest that an ion pump
be viewed (Figure 1)
as a modified ion channel with at least two gates that should never
be open simultaneously: i.e., one gate must close before the other can
open, implying tight coupling between them.
Current research
Because the Na+/K+
pump and CFTR Cl- channel both move charged ions across the
cell membrane, they generate tiny electrical signals that we can measure
to monitor their function. Using the sensitive "patch-clamp" recording
technique we can readily see the ~107 Cl- ions s-1
that flow across the membrane when a single CFTR Cl- channel
opens. Intriguingly, each CFTR molecule includes two highly conserved,
cytoplasmic, nucleotide binding domains that place it in the large family
of ABC (ATP Binding Cassette) proteins, and that are believed to sequentially
bind and hydrolyze ATP. By analyzing the timing of opening and closing
of individual wild-type or mutated CFTR channels during exposure to ATP
and/or nucleotide analogs, we are learning how the free energy released
during ATP binding and hydrolysis is harnessed to drive the conformational
changes that open and close the ion pore (Figure 2). We find that both nucleotide
binding domains must bind ATP before the channel opens, that the two domains
interact to regulate each other's function, and that one acts like a G
protein in that nucleotide bound there stabilizes the active (open) state
until hydrolysis prompts channel closure. The orders-of-magnitude smaller
ion movements generated by pumps preclude similar electrical assays of
individual Na+/K+ pump function, but we can monitor
ensemble activity of thousands of Na+/K+ pumps in
small patches of membrane, or of the millions of Na+/K+
pumps in entire cells. Our high-speed electrical measurements suggest
that, at least in the conformation that releases the 3 Na+
to the exterior, the Na+/K+ pump resembles an ion-channel
pore closed at its cytoplasmic end. That the Na+/K+
pump does contain an ion channel gated at either end is supported by our
latest work using the marine toxin, palytoxin (PTX). PTX effectively disrupts
the coupling between the Na+/K+ pump's two gates,
so permitting dissipative cation flow fast enough (~107 ions
s-1) to be measured in a single Na+/K+
pump. This ion flow is interrupted by occasional closures of one or the
other gate. Binding of external K+ tends to close, and external
Na+ to open, the extracellular gate, whereas nucleotides binding
from the cytoplasmic solution tend to open the intracellular gate. Thus,
PTX transforms the Na+/K+ pump into a channel, in
which gating seems to reflect the normal ion occlusion/deocclusion reactions
that alternately entrap first 3 Na+, and then 2 K+,
within the interior of the pump, between the two gates.
Future Goals
We are presently using site-specific mutagenesis combined
with biochemical, electrical, and fluorescence measurements to probe the
relationships between molecular structure and mechanism in these two ion
transport proteins, often one molecule at a time.
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