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The mechanism of Na-uptake by the epithelium of frog skin
Bioebctrochenzistry
am-Z Bioe?teugetics
3, 365-369
(1976)
The Mechanism of Na-Uptake by the Epithelium of Frog Skin * by B. Institute
LISDEJIASN
of Physiology
Frog
(II)
of the Saarland
skin epithelium
University,
Homburg/Saar,
F.R.G.
consists of 4 to 3 cellular layers which can, if
necessary, solve the task of transepithelial Na+-uptake by teamwork. Recent evidence has shown that their cytosols are functionally conrLfzx?EdgC&ELM_.Jr8T .zaJg :&.EtCC&Es x_CKzt%rE5 ZCKUCa CcLaLmrIUtiaEqKn2 compartment. Na+ eUnL* this compartment from the ix&i& (the sul3XK-
neal space), distributes within the cells by diffusion and is then.pumped into the interstitial space. From here it diffuses into the subepithelial tissue. However. under conditions of uninhibited active transport, the jointed pumping capacity of all cellular layers is not needed because the pumps of the outermost living cellular layer can already handle all the Na+ which enters the cytosol from the outside. In this case the transport compartment is effectively limited to the outermost cellular layer, which is called the outer Str. gvn~zz~Zos~l~~a.~-~ In any case, the first step of Na+uptake is the entrace of Na+ ions from the subcorneal space into the cytosol of the outer Stv. grnurrlosunt. This transport step, which is ratelimiting for the overall uptake and controlled by several hormones, has been the object of our investigations. The apical membrane of the Str. grmtdoszmc contains Na+-specific We have attempted channels of previously unknown physical nature. to study these channels by recording their current-voltage curve, their current response to sudden changes of Na+-concentration, their response to several chemical agents which either open or close the channels and their spontaneous current fluctuations. In these experiments the so called latero-basal membranes -of the epithelium, which face the intercellular space and have a high I<+-permeability. were depolarized by increasing the K-+-activity of the inner bathin, a solution to 60 mM. This is espected to increase the conductance of the membrane up to 5 fold and to reduce its membrane potential to values close to zero mV. The Na+ selective membrane will then dominate the electrical properties of the epithelium, its membrane voltage being equal to the transepithelial voltage within a few mV. The transepithelial voltage could therefore be used as the input signal of our voltage clamp. Paracellular currents were corrected for bk * Short Symposium
version
of the
plenary
on Bioelectrochemistry.
lecture
read
Jiilich 27-31
at
the
October
3rd 1975.
International
366
Lindemann
recording also in the presence of high concentrations of amiloride. a drug known to block the Na+-channels reversibly. Non-permeating anions like SO A”- were used throughout, and the species of frogs used was R. esc&xtn_ The amiloride-blockable current (1~~) flowing through the Natchannels x-as found to change with membrane voltage as predicted by the constant field equation-d Its rectification can therefore be esplained b)- asymmetry of Na+-acti\-ities on the two sides of the membrane, and the transport mechanism may be free electrodiffusion through waterfilled pores.” The constant field equation can thus be used to compute approsimate \-alues of permeability (E&J as well as cellular Na+-acti\*ity ([Na+]J online once e\-er:,-second, while parameters like the outside Na+concentration ([Na+],) are rapidly changed.6 A step increase of [Na+& was found to cause a rapid increase of 1~~ followed by a delayed decrease to an intermediate value.’ Computations showed that the current decrease is caused by a decrease of PN= which occurs with a relasation time constant of L-Z s. In this time [Nat]= reThe fact that ‘in such esperiments 1~~ is mains practically constant.” transiently larger than in the subsequent steady state escludes the possibility that the Na+-dependent permeability- decrease is due to a saturation of transport channels with diffusing Na+-ions. Rather. the overshooting time course of 1~ has the kinetic characteristics of substrate 192hibitioiz.. requirin g a negative effector action of Na+ which may occur outside of the transport channel itself. The permeability-modify-in, m mechanism was found to be characterized by the steady state equation
in which P,,, and KX are constants. It is eas>- to show that this dependence esplains the well-known sntzrratiox of the steady state Na+ current or Na+ flus with increasing [Na+&,_ S-II The constant field equation for V = o and [Na+j,, 9 ENa+& may be written as
where F is FARADA\-‘S constant. u-e obtain
Introduction
will
then
By substitution of PN~ using equation
(I)
of the abbreviation
J’ield the
usual
type equation which the current or flus saturation.
~IICH_IELIS--MESTES
usecl in the 1iteraturesrlO to describe
is
Xa-Uptake
by
the Epithelium
of Frog Skin_.
367
The dependence of PN~ on [Na+], can tentatively be explained by postulating a transport modifyin, m center in the vicinitv of each Na+channel. LVhen a site of this hypothetical center is occupied by a Na+, a conformational change of membrane proteins may close the adjacent channel. Alternatively, the binding of Na+ might occur in the outer opening of the transport channel, in such a way that the selectivity filter of the channel is obstructed, while Nat is bound_ In. this case KN would be the dissociation constant of Na+ at the binding site. In any case models for Nae-dependent channel obstruction require rather. low transition probabilities to esplain the lon,m relasation times of PN~, which are in the order of seconds. The decrease of PNa with increasing fNa+]b can partly or completely be prevented by the presence of drugs like benzimidazolyl-guanidinium I2 or parachlormercuribenzoate (PCMB)13 in the outer bathi_ng soEEt!. - The kinetics of these drug-effects are consistent with the hypothesis that BIG and PCMB attach themselves to the outer surface of the membrane in the vicinity of the modifying site and thus prevent binding of Na+ to this site Irreversible closure of Na+-channels can be achieved by treating the outer membrane surface with the carbosyl-blocker carbodiimide.14 Reversible closure is observed during esposure to low pHl4 or while amiloride is present in the outer solution_ It is interesting that the dose VS. response curve of amiloride shifts to higher concentrations when [Na+&, is increased, as would be espected from I : I competition betveen Na+ and amiloride. 15 In terms of models for Nae-dependent pore-occlusion this observation means that pore blockage by Na+ and amiloride is mutually esclusive. The reversibility of the amiloride effect niiy be used to ‘estimate the conductance and the Na+-turnover of single-channels and the density of channels by fluctuation analysis. A reversible blocker can be espected to randomly interrupt the Na+-turnover of individual channels; and thereby produce small current pulses of amplitude i, which add up to a mean Na+-current .
INa =
i IV, PO
(4)
Here lV-4 is the number of those amiloride blockable channels per cm’ which are not bloched by Nat. The steady state probability J?G represents the fraction of ‘AT, which is not blocked .by amiloride and ?V_&‘” the mean density of open channels. A fluctuation analysis of Na+-current in the p&e&e of amiloride was recently carried out. 16 It was found that with submasimal amiloride concentrations the power spectrum of 1~~ is of theL OREXTZ--type. Corner frequencies increased linearly with amiloride concentration. The mean lifetime of a bldcked channel was found to be in the order fo ioo ms, while the rate constant for attachment of amiloride IO per second and ii~Mel/l at 60 mM [Na+],.
to the pores ranged around
The apparent dissociation of the channel-amiloride comples decreased with decreasing [Na+lo, as espected if Na+ and amiloride are competitive -blockers. constant
Lindemann
368
At 60 m&I [Natlo the mean net Na-current through single open This corresponds to (1-3) x 106 Na+ ions s-1. channels was 0.3-0.5 pA. Thus turnover-numbers are much larger than the values of about 104 Ions s-l which are expected17 and were observed18 for carrier transport. We feel justified, therefore, in concluding that in the Na+-selective membrane of frog skin transport occurs through pores. At 60 m&f [Nay]], single channel conductances of s-10 pS were computed_ Like the smgle channel currents these values decreased with decreasing [Na+]?. The mean permeability of single open channels was estimated to be m the order of 10-l~ cm3 per s per channel area. The combined density of open pores plus amiloride-blocked pores was found to be in the range (o -7-z) x Io*/cm% of membrane area at 60 m.M [Na+],,_ When [Na+],, was lowered to 15 mM by substitution with K+, Iarger pore densities were computed, which. however, did not esceed 3 x 10~ pores/cm”-. pores with decreasing [Na+], is This increase in “amiloride-blockable” to be espected if Na and amiloride are competitive blockers. Extrapolation to [Na+J-, = o shows that the total number of Na+-translocating channels will be below 5 x 10~ per cm= of epithelial area. This density corresponds to 50 pores per pm2 membrane area if a homogeneous distribution of Dares over the aDica1 membranes of ail cells of the outer Stv. gmnztloszc$may- be assumGd_
Acknowledgements This research was supported by the Deutsche Forschungsgemeinschaft through SFB $5, Project C I.
References 1
6
7
8
A. DIRGE, R. RICK, S. I~ELSOS, R. BAUER and K. THURAU. Pflzre,oevs Arcir. 362, R 14 (1~76) F. MOREL and G. LEELASC. Pfhregevs AYC~. 358, 135 (r975) CL. VO~~TE and H-H. USSISG, J_ Cell BioE. 36, 625 (1~65) XL. HODGKIS and B. KATZ. J. Physiol. 108, 37 (1949) \V. FUCHS, E. HVIID LARSES and B. LIXDEMASS, PfZzregers AYCJZ. 355, R 71 (1973) B. CIRSE and B. LISDEUXSS. PnssitleIVatrsre of Li+ and Na+ Tramport Biophysics thvorr,ok tke Na-SeZeciive Menzbrarze of Frog Skix. 5th International Congress, P 121. Copenhagen (1975) B. LIXDEMASS and U. GEBHXRDT in TvansPort Mecl~amhns in Epitheiia. H.H. USSISG and X-X. THORS (Editors), Munksgaard, Copenhagen and Academic Press, X.T. (1973) pp. IIS-130. T.U.L. BIBER and P.F. CURRAS, J_ Gen. PI~ysioZ.56, 83 (1970)
Na-Uptake
9 10
11 12
M.
CERELJIDO.
by
F.C. HERRERA,
Physiol. 47, 379 (1964) D. ERLIJ and M.W. SMITH, LA.
SALXKO
13 14
W. ZEISKE H.J. DICK IV. ZEISKE
15
A.W.
the
and-A-J.
Epithelium
W.J.
FLAKIGAX
J_ PhysioZ.
SMITH,
of
228,
ZZI
BY. J. Pha~nzacoZ.
Frog and
17 18
J_
Gen.
and
B.
LIKDEMASX,
Biochim.
Bio$hys_
B.
LINDEMAX~~-,
PfIrregers
Arch.
and
B. LISDE'JIANX.
Pfluegers Nazrnyn
Arch. 355, R7z (1975) Schtniedebevg’s Arch. PhaymacoZ.
and
DRIESSCHE
C.&I. ARMSTRONG, P. LXUGER.
CURRAX,
and
CUTHBERT
VAN
P.F.
369
(rg73) 39, gg (rg7o)
\V.K_
SHUM.
and Biophys.
B. LISDEMANN. J_
15,
Science 178, 24 (1972)
932
Acfa
355,
281, 261 (w7.4 16 .\V.
Skin.
Pfiuegers (1975)
352, 323 (1974) -R-72 (1975)
Arch..
362,
R 28
(1976)
am-Z Bioe?teugetics
3, 365-369
(1976)
The Mechanism of Na-Uptake by the Epithelium of Frog Skin * by B. Institute
LISDEJIASN
of Physiology
Frog
(II)
of the Saarland
skin epithelium
University,
Homburg/Saar,
F.R.G.
consists of 4 to 3 cellular layers which can, if
necessary, solve the task of transepithelial Na+-uptake by teamwork. Recent evidence has shown that their cytosols are functionally conrLfzx?EdgC&ELM_.Jr8T .zaJg :&.EtCC&Es x_CKzt%rE5 ZCKUCa CcLaLmrIUtiaEqKn2 compartment. Na+ eUnL* this compartment from the ix&i& (the sul3XK-
neal space), distributes within the cells by diffusion and is then.pumped into the interstitial space. From here it diffuses into the subepithelial tissue. However. under conditions of uninhibited active transport, the jointed pumping capacity of all cellular layers is not needed because the pumps of the outermost living cellular layer can already handle all the Na+ which enters the cytosol from the outside. In this case the transport compartment is effectively limited to the outermost cellular layer, which is called the outer Str. gvn~zz~Zos~l~~a.~-~ In any case, the first step of Na+uptake is the entrace of Na+ ions from the subcorneal space into the cytosol of the outer Stv. grnurrlosunt. This transport step, which is ratelimiting for the overall uptake and controlled by several hormones, has been the object of our investigations. The apical membrane of the Str. grmtdoszmc contains Na+-specific We have attempted channels of previously unknown physical nature. to study these channels by recording their current-voltage curve, their current response to sudden changes of Na+-concentration, their response to several chemical agents which either open or close the channels and their spontaneous current fluctuations. In these experiments the so called latero-basal membranes -of the epithelium, which face the intercellular space and have a high I<+-permeability. were depolarized by increasing the K-+-activity of the inner bathin, a solution to 60 mM. This is espected to increase the conductance of the membrane up to 5 fold and to reduce its membrane potential to values close to zero mV. The Na+ selective membrane will then dominate the electrical properties of the epithelium, its membrane voltage being equal to the transepithelial voltage within a few mV. The transepithelial voltage could therefore be used as the input signal of our voltage clamp. Paracellular currents were corrected for bk * Short Symposium
version
of the
plenary
on Bioelectrochemistry.
lecture
read
Jiilich 27-31
at
the
October
3rd 1975.
International
366
Lindemann
recording also in the presence of high concentrations of amiloride. a drug known to block the Na+-channels reversibly. Non-permeating anions like SO A”- were used throughout, and the species of frogs used was R. esc&xtn_ The amiloride-blockable current (1~~) flowing through the Natchannels x-as found to change with membrane voltage as predicted by the constant field equation-d Its rectification can therefore be esplained b)- asymmetry of Na+-acti\-ities on the two sides of the membrane, and the transport mechanism may be free electrodiffusion through waterfilled pores.” The constant field equation can thus be used to compute approsimate \-alues of permeability (E&J as well as cellular Na+-acti\*ity ([Na+]J online once e\-er:,-second, while parameters like the outside Na+concentration ([Na+],) are rapidly changed.6 A step increase of [Na+& was found to cause a rapid increase of 1~~ followed by a delayed decrease to an intermediate value.’ Computations showed that the current decrease is caused by a decrease of PN= which occurs with a relasation time constant of L-Z s. In this time [Nat]= reThe fact that ‘in such esperiments 1~~ is mains practically constant.” transiently larger than in the subsequent steady state escludes the possibility that the Na+-dependent permeability- decrease is due to a saturation of transport channels with diffusing Na+-ions. Rather. the overshooting time course of 1~ has the kinetic characteristics of substrate 192hibitioiz.. requirin g a negative effector action of Na+ which may occur outside of the transport channel itself. The permeability-modify-in, m mechanism was found to be characterized by the steady state equation
in which P,,, and KX are constants. It is eas>- to show that this dependence esplains the well-known sntzrratiox of the steady state Na+ current or Na+ flus with increasing [Na+&,_ S-II The constant field equation for V = o and [Na+j,, 9 ENa+& may be written as
where F is FARADA\-‘S constant. u-e obtain
Introduction
will
then
By substitution of PN~ using equation
(I)
of the abbreviation
J’ield the
usual
type equation which the current or flus saturation.
~IICH_IELIS--MESTES
usecl in the 1iteraturesrlO to describe
is
Xa-Uptake
by
the Epithelium
of Frog Skin_.
367
The dependence of PN~ on [Na+], can tentatively be explained by postulating a transport modifyin, m center in the vicinitv of each Na+channel. LVhen a site of this hypothetical center is occupied by a Na+, a conformational change of membrane proteins may close the adjacent channel. Alternatively, the binding of Na+ might occur in the outer opening of the transport channel, in such a way that the selectivity filter of the channel is obstructed, while Nat is bound_ In. this case KN would be the dissociation constant of Na+ at the binding site. In any case models for Nae-dependent channel obstruction require rather. low transition probabilities to esplain the lon,m relasation times of PN~, which are in the order of seconds. The decrease of PNa with increasing fNa+]b can partly or completely be prevented by the presence of drugs like benzimidazolyl-guanidinium I2 or parachlormercuribenzoate (PCMB)13 in the outer bathi_ng soEEt!. - The kinetics of these drug-effects are consistent with the hypothesis that BIG and PCMB attach themselves to the outer surface of the membrane in the vicinity of the modifying site and thus prevent binding of Na+ to this site Irreversible closure of Na+-channels can be achieved by treating the outer membrane surface with the carbosyl-blocker carbodiimide.14 Reversible closure is observed during esposure to low pHl4 or while amiloride is present in the outer solution_ It is interesting that the dose VS. response curve of amiloride shifts to higher concentrations when [Na+&, is increased, as would be espected from I : I competition betveen Na+ and amiloride. 15 In terms of models for Nae-dependent pore-occlusion this observation means that pore blockage by Na+ and amiloride is mutually esclusive. The reversibility of the amiloride effect niiy be used to ‘estimate the conductance and the Na+-turnover of single-channels and the density of channels by fluctuation analysis. A reversible blocker can be espected to randomly interrupt the Na+-turnover of individual channels; and thereby produce small current pulses of amplitude i, which add up to a mean Na+-current .
INa =
i IV, PO
(4)
Here lV-4 is the number of those amiloride blockable channels per cm’ which are not bloched by Nat. The steady state probability J?G represents the fraction of ‘AT, which is not blocked .by amiloride and ?V_&‘” the mean density of open channels. A fluctuation analysis of Na+-current in the p&e&e of amiloride was recently carried out. 16 It was found that with submasimal amiloride concentrations the power spectrum of 1~~ is of theL OREXTZ--type. Corner frequencies increased linearly with amiloride concentration. The mean lifetime of a bldcked channel was found to be in the order fo ioo ms, while the rate constant for attachment of amiloride IO per second and ii~Mel/l at 60 mM [Na+],.
to the pores ranged around
The apparent dissociation of the channel-amiloride comples decreased with decreasing [Na+lo, as espected if Na+ and amiloride are competitive -blockers. constant
Lindemann
368
At 60 m&I [Natlo the mean net Na-current through single open This corresponds to (1-3) x 106 Na+ ions s-1. channels was 0.3-0.5 pA. Thus turnover-numbers are much larger than the values of about 104 Ions s-l which are expected17 and were observed18 for carrier transport. We feel justified, therefore, in concluding that in the Na+-selective membrane of frog skin transport occurs through pores. At 60 m&f [Nay]], single channel conductances of s-10 pS were computed_ Like the smgle channel currents these values decreased with decreasing [Na+]?. The mean permeability of single open channels was estimated to be m the order of 10-l~ cm3 per s per channel area. The combined density of open pores plus amiloride-blocked pores was found to be in the range (o -7-z) x Io*/cm% of membrane area at 60 m.M [Na+],,_ When [Na+],, was lowered to 15 mM by substitution with K+, Iarger pore densities were computed, which. however, did not esceed 3 x 10~ pores/cm”-. pores with decreasing [Na+], is This increase in “amiloride-blockable” to be espected if Na and amiloride are competitive blockers. Extrapolation to [Na+J-, = o shows that the total number of Na+-translocating channels will be below 5 x 10~ per cm= of epithelial area. This density corresponds to 50 pores per pm2 membrane area if a homogeneous distribution of Dares over the aDica1 membranes of ail cells of the outer Stv. gmnztloszc$may- be assumGd_
Acknowledgements This research was supported by the Deutsche Forschungsgemeinschaft through SFB $5, Project C I.
References 1
6
7
8
A. DIRGE, R. RICK, S. I~ELSOS, R. BAUER and K. THURAU. Pflzre,oevs Arcir. 362, R 14 (1~76) F. MOREL and G. LEELASC. Pfhregevs AYC~. 358, 135 (r975) CL. VO~~TE and H-H. USSISG, J_ Cell BioE. 36, 625 (1~65) XL. HODGKIS and B. KATZ. J. Physiol. 108, 37 (1949) \V. FUCHS, E. HVIID LARSES and B. LIXDEMASS, PfZzregers AYCJZ. 355, R 71 (1973) B. CIRSE and B. LISDEUXSS. PnssitleIVatrsre of Li+ and Na+ Tramport Biophysics thvorr,ok tke Na-SeZeciive Menzbrarze of Frog Skix. 5th International Congress, P 121. Copenhagen (1975) B. LIXDEMASS and U. GEBHXRDT in TvansPort Mecl~amhns in Epitheiia. H.H. USSISG and X-X. THORS (Editors), Munksgaard, Copenhagen and Academic Press, X.T. (1973) pp. IIS-130. T.U.L. BIBER and P.F. CURRAS, J_ Gen. PI~ysioZ.56, 83 (1970)
Na-Uptake
9 10
11 12
M.
CERELJIDO.
by
F.C. HERRERA,
Physiol. 47, 379 (1964) D. ERLIJ and M.W. SMITH, LA.
SALXKO
13 14
W. ZEISKE H.J. DICK IV. ZEISKE
15
A.W.
the
and-A-J.
Epithelium
W.J.
FLAKIGAX
J_ PhysioZ.
SMITH,
of
228,
ZZI
BY. J. Pha~nzacoZ.
Frog and
17 18
J_
Gen.
and
B.
LIKDEMASX,
Biochim.
Bio$hys_
B.
LINDEMAX~~-,
PfIrregers
Arch.
and
B. LISDE'JIANX.
Pfluegers Nazrnyn
Arch. 355, R7z (1975) Schtniedebevg’s Arch. PhaymacoZ.
and
DRIESSCHE
C.&I. ARMSTRONG, P. LXUGER.
CURRAX,
and
CUTHBERT
VAN
P.F.
369
(rg73) 39, gg (rg7o)
\V.K_
SHUM.
and Biophys.
B. LISDEMANN. J_
15,
Science 178, 24 (1972)
932
Acfa
355,
281, 261 (w7.4 16 .\V.
Skin.
Pfiuegers (1975)
352, 323 (1974) -R-72 (1975)
Arch..
362,
R 28
(1976)