Electrophysiological techniques are central for the investigation of ion
transport mechanisms. Our laboratory has successfully applied electrophysiological
approaches to CF research. By using the patch clamp and the Ussing chamber
techniques our investigations opened up significant new avenues for CF
This technique is used to measure ion transport
by intact, isolated epithelial tissue. It is named after the Danish physiologist
Hans Ussing who invented it. We use it for drug screening and to verify
results from other techniques. These measurements are quantitative, easy
to learn, and quick.
. This method is used to
measure epithelial acid or base secretion. It is based on the continuous
titration of the epithelial bathing medium to a given pH, which then quantifies
secretion of acid (or base) by the epithelium. Using this technique, we
have recently discovered that the airway epithelium secretes acid into
Figure 1 Patch clamping of single cells
on the stage of a microscope
. Patch clamping is
used to measure ion currents across a single cell or through a single
ion channel in the cell membrane. Cells are investigated in isolation
on the stage of a microscope. A small glass pipette (~1 mm inner diameter)
is attached onto a cell and used as an electrode. We use various patch
clamp techniques, including whole cell and cell-attached recordings, and
recordings from excised inside-out or outside-out membrane patches. In
addition we use noise analytical techniques to investigate channel-types
that are difficult to record with standard patch-clamp techniques. Patch-clamping
is extremely versatile and we use it to investigate: 1) intracellular
signaling cascades that regulate ion channel activity, 2) channel characteristics
and kinetics, and 3) drug effects on ion channels.
Recordings of the nasal potential difference (nasal PD). This is a technique
that is used to indirectly measure channel activity in people. We measure
ionselective diffusion potentials across the nasal epithelium. The nose
is used because it is easily accessible. It has been shown that the nasal
PD is significantly changed in CF. We use these measurements as a diagnostic
test for CF, and for in vivo drug testing.
One current CF-related project is the role and regulation of basolateral
chloride channels in salt secretion and absorption across airways. The
primary transport process across the airways is active sodium absorption.
Chloride follows passively according to its electrochemical driving forces.
During transcellular chloride movement chloride has to cross the apical
and the basolateral membrane. Previous work has focused on the apical
membrane where CFTR is expressed (the chloride channel that is defective
in CF). Transport across the basolateral membrane was unclear. We found
that three physiologically and biophysically distinct chloride channels
are present and functional, and they determine the overall rate of chloride
transport by the epithelium.
Another project is the acid and base transport across the airway epithelium.
Interestingly, the airway surface liquid (ASL) is slightly acidic (pH
= 6.9) compared to other extracellular fluids (pH = 7.4) in normals. It
has been hypothesized that ASL in both CF and asthma is even more acidic
than normal. The source of the acidity is unknown. We hypothesized that
the airway epithelium expresses apical proton transporters, which acidify
the ASL in a regulated manner. In a study using the pH stat technique
and patch clamping we found apical proton channels in airway epithelial
cells, which are the major route for acid secretion into the ASL. Currently
we investigate whether the proton channels are mis-regulated in asthma
and CF. In addition we investigate the secretion of bicarbonate (which
buffers protons in the ASL). Our results suggest that a disequilibrium
between acid (proton) and base (bicarbonate) secretion into the ASL is
an important factor in the pathogenesis of CF and asthma.