ABBS 2005,38(02): Kir6.2DC26 Channel Traffics to Plasma Membrane by Constitutive Exocytosis

*Corresponding author: Tel, 86-27-87792024; Fax, 86-27-87792024; E-mail,
[email protected]

Abstract        Adenosine triphosphate (ATP)-sensitive
K+ (KATP) channels regulate many cellular functions by
coupling the metabolic state of the cell to the changes in membrane potential.
Truncation of C-terminal 26 amino acid residues of Kir6.2 protein (Kir6.2DC26) deletes its endoplasmic reticulum retention
signal, allowing functional expression of Kir

Key words        Kir6.2D26; exocytosis;
whole-cell current; total internal reflection fluorescence microscopy; patch
clamp

Adenosine triphosphate
(ATP)-sensitive K+ (KATP) channels are found in a variety of tissues,
including pancreatic b-cells and
central neurons, as well as cardiac, smooth and skeletal muscle cells [1–3]. They regulate many cellular functions by coupling the metabolic state
of the cell to electrophysiological membrane potential [4]. The KATP channel in the plasma membrane consist of four
inwardly rectifying potassium channel (Kir) a subunits (Kir6.1/6.2) and four regulatory sulphonylurea receptor (SUR) b subunits
(SUR1/Using epi-fluorescence
and total internal reflection fluorescence microscopy (TIRFM), we investigated
the cellular distribution and trafficking mechanisms of enhanced green
fluorescent protein (EGFP)-Kir6.2DC26 fusion
protein expressed in HEK293 cells. Our data showed that EGFP-Kir6.2DC26 fusion proteins are mainly localized on the
plasma membrane and trafficked and incorporated into the plasmalemma by
constitutive exocytosis of Kir6.2DC26 transport
vesicles (KTVs). We further examined the functional manifestation of Kir6.2DC26 by measurement of whole-cell currents. To
exclude the possibility that the tagged EGFP might alter the physiological
function of fusion proteins [13,14], we constructed an internal ribosome entry site
(IRES), which will truncate translation of Kir6.2DC26 and reinitiate translation of EGFP to let two
proteins be expressed simultaneously and separately, into pKir6.2DC26-IRES2-EGFP vector. Cells transfected with
pKir6.2DC26-IRES2-EGFP
vector were used for electrophysiological experiments. The ATP-sensitive
currents of Kir6.2DC26
demonstrated that the Kir6.2DC26 protein
complex in the plasma membrane expresses KATP channel activity. These two lines of evidence,
which supplement each other, suggest that Kir6.2DC26 is a good tool for genetic manipulation, and for
studying the structure and function of the Kir6.2 channel and even its
regulation of trafficking.­­­­­­­

Materials and Methods

Construction of
pEGFP-Kir6.2D26 and
pKir6.2D26-IRES2-EGFP
plasmids

The cDNA of Kir6.2DC26 was amplified by polymerase chain reaction (PCR)
using PfuUltra high-fidelity DNA polymerase (Clontech, Palo Alto, USA),
with a template of mouse brain marathon-ready cDNA (Clontech). The sequences of
Kir6.2DC26 primers were:
forward, 5‘-TACCACCATGCATCATCACCATCACCATATCGAAGGT­AGGATGCTGTCCCGAAAGGGCATTATCC-3‘
and reverse, 5‘-TTATCACGACGAGGCGAGGGTCAGGGCATCCAGCAGACTGCGGTCCTC-3‘.
The PCR product was subcloned into the vector pCR2.1-TOPO (Invitrogen,

Cell culture and
transfection

HEK293 cells were kindly
provided by Dr. David Zhi-Qing XU (Karolinska Institute, Stockholm, Sweden) and
grown in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, USA) supplemented
with 10% fetal bovine serum (Gibco) at 37 ºC with a humidified mixture of 5% CO2 and 95% air. The cells were passaged at a
ratio of approximately 1:3 by treatment of trypsin/EDTA once per week. Cells
cultured for 2–4 d were
transfected for 48 h using the recombinant plasmids pKir6.2DC26-IRES2-EGFP and pEGFP-Kir6.2DC26 with an Effectene transfection reagent kit
(Qiagen,

Fluorescence imaging and
data analysis

The epi-fluorescence and
TIRFM system were constructed as described previously [15]. Briefly,
fluorescent subcellular structures were imaged by a 100´  Apo OHR
objective lens (NA 1.65; Vesicles that underwent
exocytosis were visually selected. We put a small square (1.14 mm´1.14 mm) and a big square (2.35 mm´2.35 mm) on the fusing vesicle co-centrad. The average
intensity of the fluorescence in the square annulus between two concentric
squares was counted as the local background and subtracted for each vesicle. We
then used two concentric circles (13 and 17 pixels with a pixel size of 67 nm,
corresponding to approximately 0.87 mm and
approximately 1.14 mm in
diameter), centered on a selected vesicle, to characterize the fluorescence
changes of the single vesicle in background-subtracted images. The mean
brightness of the inner circle was used for measuring fluorescence intensity of
the individual vesicle. Simultaneously, the average fluorescence in the
concentric annulus was calculated as a marker for identifying the diffusion of
Kir6.2DC26-EGFP
fusion proteins from the inner circle to the outer circle.

Whole-cell current
measurement

All electrophysiological
experiments were carried out on HEK293 cells at room temperature (22–25 ºC). Whole-cell currents were recorded with an
EPC9 amplifier (Heka Electronics,

Results

PCR amplification of Kir6.2DC26 cDNA
fragment

The cDNA encoding Kir6.2DC26 was amplified by PCR with a template of mouse
brain marathon-ready cDNA library. The amplified products were separated by 1%
agarose gel electrophoresis. Fig. 1 shows one specific approximately
1138 bp fragment.

Localization and
trafficking of Kir6.2D26 channels

We examined the cellular
distribution and trafficking of EGFP-Kir6.2DC26 using epi-fluorescence and TIRFM. Under an
epi-fluorescence microscope with the focus plane across the center of the cell,
the plasma membrane was brightened with green fluorescence [Fig. 2(A,C)].
The result showed that the EGFP-Kir6.2DC26 proteins
were correctly targeted and transported to the plasma membranes in HEK293 cells.
We employed the inherent advantage of highlighting the events within
approximately 100 nm of the plasma membrane with TIRFM to check and visualize
the translocation of Kir6.2DC26 channels
to the plasmalemma [15]. It is accepted that membrane proteins, including ion
channels and receptors, are transported to the plasmalemma by means of
exocytosis of protein-transporting vesicles [12]. We observed many events of
vesicle translocation, docking and fusion (data not shown). The full width at
half maximum fluorescence of isolated bright fluorescence spots by Gaussian
fitting, which represents the diameter of vesicles under TIRFM [16,17], was
322+/–5 nm (n=25), which was similar to that of the
NPY-DsRed-labeled LDCVs in PC12 cells (384+/–6 nm, n=30)
(data not shown). Therefore, we suggest that the round fluorescent spots were
KTVs [Fig. 2(B) and Fig. 3(A)].

By sequential imaging at
a frequency of 5 Hz under TIRFM, we monitored the trafficking of Kir6.2DC26 tagged with fluorescence marker EGFP to the
plasma membrane. Spontaneous fusion events of Kir6.2 transport vesicles were
observed [Fig. 3(A)]. We observed a sudden and brief brightening of the
fluorescence spot which was visible previously. Fig. 3(B) shows the
fluorescence changes of KTV shown in Fig. 3(A) versus time (the time
point at the peak of the fluorescence trace was set to zero). Luminal EGFP
tagged to the N-terminal of Kir6.2DC26 contained
in acidic granules brightens up when it encounters the neutral pH of the
external medium during fusion pore opening, therefore a sudden increase in
vesicle fluorescence is regarded as a fusion event. To exploit the dynamics of
exocytosis of KTVs and protein diffusion, we averaged the normalized
fluorescence (18 events, 5 cells), taking the time of onset of vesicle fusion
as zero. Fig. 3(C) shows the dynamics of vesicular fluorescence spots
and concentric annuli around the circles. We focused on the dynamics of
fluorescence decay. The time-course of fluorescence changes was best fitted by
double exponential function with a time constant of 0.16 s and 8.96 s for fast
(t1) and slow (t2) components,
respectively [Fig. 3(C), upper panel]. The mean fluorescent intensity of
concentric annulus displayed moderate sigmoid changes [Fig. 3(C), lower
panel].

Electrophysiological characterization
of Kir6.2D26 channels

To assay whether
truncated Kir6.2 expresses ATP-sensitive currents, we recorded whole-cell
currents in intact HEK293 cells (as a control [cont] for electrophysiological
experiments), cells transfected with pKir6.2D26-IRES2-EGFP (IK), or
with pEGFP-Kir6.2D6 (EK) using pulse
depolarization stimuli, as shown in the lower panel of Fig. 4(A). The
upper panel of Fig. 4(A) shows example traces of whole-cell currents of
these cells. The intact HEK293 cells expressed very small currents [Fig.
4(A), control, t=0] and were not sensitive to ATP, as indicated by
no significant change of currents after more than 8 min of ATP dilution with
intracellular solution containing Fig. 4(B) summarizes
the average amplitude of whole-cell currents at the time of onset of whole-cell
configuration (t=0), more than 8 min of ATP dilution by intracellular
solution containing

Discussion

Truncation of the
C-terminal of Kir6.2 deletes its ER retention signal, allowing functional
expression of KirThe KATP channel is
normally formed
as an octamer consisting of four Kir6.2 subunits that generate the pore, and
four SUR1 subunits. Kir6.2DC26 tetramers
can assemble into potassium-permitting channels in the absence of SUR1 on the
plasma membrane [18]. Whole-cell currents recorded from HEK293 cells
transfected with pEGFP-Kir6.2DC26 are not
significantly different from that of control cells and not sensitive to ATP,
indicating the tagged EGFP alters the physiological function of Kir6.2DC26, as reported in other fusion proteins [13,14].
Then we took advantage of the bicistronic pIRES2-EGFP, which contains IRES of
the encephalomyocarditis virus between the multiple cloning sites and the
EGFP-coding region, to express Kir6.2DC26 and the
fluorescence marker EGFP separately. HEK293 cells co-expressing Kir6.2DC26 and EGFP proteins express notable potassium
currents. The characteristics of channels, for example, their sensitivity to
intracellular ATP, are identified by the current increase after ATP dilution
and current decrease after ATP perfusion. Our electrophysiological data further
confirmed that Kir6.2DC26 is able
to express ATP-sensitive potassium currents in the absence of the SUR1 subunit,
as reported in Xenopus oocytes and mammalian cells such as HEK293 cells,
COS-1 cells, and CHO cells [11,19,20].

In summary, our data
demonstrate that Kir6.2DC26 protein is
correctly trafficked to the plasma membrane by means if constitutive exocytosis
of Kir6.2 transport vesicles, and express KATP currents. Kir6.2DC26 supplies a good molecular tool for genetic
manipulation of KATP channels, which allows for future detailed
biochemical and structural analysis.

References

 1   Seino S. Physiology and pathophysiology of KATP channels
in the pancreas and cardiovascular system: A review. J Diabetes Complications
2003, 17: 2–5

 2   Nichols CG, Lederer WJ. Adenosine
triphosphate-sensitive potassium channels in the cardiovascular system. Am
J Physiol 1991, 261: H1675–H1686

 3   Quayle JM, Nelson MT,  4   Yokoshiki H, Sunagawa M, Seki T, Sperelakis
N. ATP-sensitive K+ channels in pancreatic,
cardiac, and vascular smooth muscle cells. Am J Physiol 1998, 274: C25–C37

 5   Seino S, Miki T. Physiological and
pathophysiological roles of ATP-sensitive K+ channels. Prog Biophys Mol Biol 2003, 81: 133–176

 6   Matsuo M, Tucker SJ, Ashcroft FM, Amachi T,
Ueda K. NEM modification prevents high-affinity ATP binding to the first
nucleotide binding fold of the sulphonylurea receptor, SUR1. FEBS Lett 1999,
458: 292–294

 7   Ma D, Jan LY. ER transport signals and
trafficking of potassium channels and receptors. Curr Opin Neurobiol 2002, 12:
287–292

 8   Zerangue N, Schwappach B, Jan YN, Jan LY. A
new ER trafficking signal regulates the subunit stoichiometry of plasma
membrane KATP channels.
Neuron 1999, 22: 537–548

 9   Gribble FM, Ashfield R, Ammala C, Ashcroft
FM. Properties of cloned ATP-sensitive K+ currents expressed in Xenopus oocytes.
J Physiol 1997, 498: 87–98

10  Inagaki N, Gonoi T, Clement JP, Wang CZ,
Aguilar-Bryan L, Bryan J, Seino S. A family of sulfonylurea receptors
determines the pharmacological properties of ATP-sensitive K+ channels. Neuron 1996, 16: 1011–1017

11  Tucker SJ, Gribble FM, Zhao C, Trapp S,
Ashcroft FM. Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea
receptor. Nature 1997, 387: 179–183

12  Heusser K, Schwappach B. Trafficking of
potassium channels. Curr Opin Neurobiol 2005, 15: 364–369

13  Bukauskas FF, Jordan K, Bukauskiene A, Bennett
MV, Lampe PD, Laird DW, Verselis VK. Clustering of connexin 43-enhanced green
fluorescent protein gap junction channels and functional coupling in living
cells. Proc Natl Acad Sci USA 2000, 97: 2556–2561

14  Beltramello M, Bicego M, Piazza V, Ciubotaru
CD, Mammano F, D’Andrea P. Permeability and gating properties of human
connexins 26 and 30 expressed in HeLa cells. Biochem Biophys Res Commun
2003, 305: 1024–1033

15  Wu ZX, Xia S, Xu L, Bai L, Xu T. Dynamic
imaging of single secretory granule in cultured PC12 cells. Acta Biochim
Biophys Sin 2003, 35: 381–386

16  Rohrbach A. Observing secretory granules with
a multiangle evanescent wave microscope. Biophys J 2000, 78: 2641–2654

17  Tang Q, Edidin M. Vesicle trafficking and cell
surface membrane patchiness. Biophys J 2001, 81: 196–203

18  Loussouarn G, Makhina EN, Rose T, Nichols CG.
Structure and dynamics of the pore of inwardly rectifying KATP channels. J Biol Chem 2000, 275: 1137–1144

19 Sunaga Y, Gonoi T, Shibasaki T, 20  Babenko AP, Gonzalez G, Aguilar-Bryan L, Bryan
J. Reconstituted human cardiac KATP channels: Functional identity with the native
channels from the sarcolemma of human ventricular cells. Circ Res 1998,
83: 1132–1143