Permeant Anions Control Gating of Calcium-dependent Chloride Channels
P. Perez-Cornejo
1,3
, J.A. De Santiago
2
, J. Arreola
2,3
1
School of Medicine, Universidad Autonoma de San Luis Potosi, 78290 Mexico
2
Institute of Physics, Universidad Autonoma de San Luis Potosi, 78290 Mexico
3
Center for Oral Biology, University of Rochester, Medical Center, Rochester, NY 14642, USA
Received: 30 October 2003/Revised: 13 February 2004
Abstract. The effects of external anions (SCN
)
,
NO
À
3
,I
)
,Br
)
,F
)
, glutamate, and aspartate) on gat-
ing of Ca
2+
-dependent Cl
)
channels from rat parotid
acinar cells were studied using the whole-cell config-
uration of the patch-clamp technique. Shifts in the
reversal potential of the current induced by
replacement of external Cl
)
with foreign anions, gave
the following selectivity sequence based on permea-
bility ratios (P
x
/P
Cl
): SCN
)
>I
)
>NO
À
3
>Br
)
>Cl
)
>
F
)
>aspartate>glutamate. Using a continuum elec-
trostatic model we calculated that this lyotropic se-
quence resulted from the interaction between anions
and a polarizable tunnel with an effective dielectric
constant of $23. Our data revealed that anions with
P
x
/P
Cl
> 1 accelerated activation kinetics in a volt-
age-independent manner and slowed deactivation
kinetics. Moreover, permeant anions enhanced
whole-cell conductance (g, an index of the apparent
open probability) in a voltage-dependent manner,
and shifted leftward the membrane potential-g
curves. All of these effects were produced by the an-
ions with an effectiveness that followed the selectivity
sequence. To explain the effects of permeant anions
on activation kinetics and g
Cl
we propose that there
are 2 different anion-binding sites in the channel. One
site is located outside the electrical field and controls
channel activation kinetics, while a second site is lo-
cated within the pore and controls whole-cell con-
ductance. Thus, interactions of permeant anions with
these two sites hinder the closing mechanism and
stabilize the channel in the open state.
Key words: Ca
2+
-dependent Cl
)
channels — Gating
— Anion selectivity — Acinar cells — Permeation
Introduction
It is thought that a rapid Cl
)
loss to the luminal side
of salivary gland acinar cells is the critical event in the
signaling cascade triggered by muscarinic stimulation
leading to fluid secretion (Turner, 1993; Cook et al.,
1994). In rat parotid glands, Ca
2+
-dependent Cl
)
channels appear to be the exit pathway for Cl
)
ions
driving fluid secretion (Arreola, Melvin & Begenisich,
1996a; Begenisich & Melvin, 1998; Kidd & Thorn,
2000). Localization of these channels to the apical
membrane in acinar cells (Tan, Marty & Trautmann,
1992) suggests that they are exposed to large changes
in both extracellular and intracellular [Cl
)
] during the
active phase of secretion and thus are subject to
regulation by the permeant anion itself.
Recent evidence suggests that foreign permeant
anions and changes in [Cl
)
] affect Ca
2+
-dependent
Cl
)
channels present in several cells. In these channels
a prominent effect observed by replacing Cl
)
with
anions like SCN
)
,I
)
,NO
À
3
,orBr
)
, is a speeding of
the activation kinetics (Evans & Marty, 1986; Ish-
ikawa & Cook, 1993). This effect can also be ob-
served by increasing the internal Cl
)
concentration
(Pappone & Lee, 1995). On the other hand, Green-
wood and Large (1999) reported that in smooth
muscle cells, foreign anions slow the decay of Ca
2+
-
dependent Cl
)
currents in a voltage-independent
manner. The authors suggested that this alteration
was brought about by binding of the anions to a site
accessible from the external surface of the membrane.
In addition, effects of permeant anions on gating
have also been documented in ClC-0, ClC-1 and ClC-
2, all voltage-gated Cl
)
channels belonging to the ClC
Cl
)
channel family (Dinudom, Young & Cook, 1993;
Chen & Miller, 1996; Pusch et al., 1995, 1999; Fahlke,
Du
¨
rr & George, 1997; Rychkov et al., 1998). Anion
occupancy of the pore has been linked to fast gating
in ClC chloride channels (Chen & Miller, 1996; Pusch
Correspondence to: J. Arreola; email: arreola@dec1.ifisica.uaslp.mx
J. Membrane Biol. 198, 125–133 (2004)
DOI: 10.1007/s00232-004-0659-x
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