CHARACTERISATION OF A SUB-PICOSIEMENS K⁺ PERMEABILITY IN THE CRYPT BASOLATERAL MEMBRANE.

9.1. INTRODUCTION.

Whole-cell recordings have indicated that the resting membrane potential of small intestinal crypts is dominated by a resting K⁺ conductance. Single channel recordings obtained with Na gluconate or KCl-rich pipette solutions have not uncovered the identity of the single channel activity underlying the resting membrane K⁺ conductance. Although strongly inwardly-rectifying single channel activity present in cell-attached patches is spontaneously active, the inward rectification of this channel and its inactivation at depolarised holding potentials does not correlate with the outwardly rectifying whole-cell currents observed in the unstimulated crypt. These observations raise the possibility that such a conductance may be too small to resolve at the single channel level.

Ion substitution experiments were performed upon excised inside-out basolateral membrane patches, obtained with Na gluconate-rich pipette solutions to establish the ionic permeability of the membrane patch. The data obtained from such experiments is consistent with the presence of a K⁺-selective cationic permeability pathway in the crypt basolateral membrane. It is proposed that this cationic conductance underlies the basolateral K⁺ conductance which dominates the resting membrane potential of small intestinal crypts.

9.2. METHODS.

Small intestinal crypts were prepared using the high K⁺ isolation method described previously in section 6.2.1.

9.2.1. SOLUTIONS.

The concentrations of Ca²⁺ and EGTA required to buffer `cytosolic' free Ca²⁺ in the bath to 36nM and 1m M were calculated according to the stability constants of Martell & Smith (1974). Low K⁺ pipette solutions were prepared from 1M KCl stock solutions. A 1M stock solution of N-methyl D-glucamine Cl was prepared by dissolving the appropriate quantity of N-methyl D-glucamine in hydrochloric acid. The composition of all solutions is given in table 9.1.

9.2.2. Electrophysiological recordings.

Inside-out patches were formed using the methodology described in 8.2.2. with the following modifications. After fire-polishing the tips had resistances of 8-10 MW when back-filled with a Na Gluconate rich solution (except experiments performed with 0.1mM K⁺ where pipette resistances of 10-20 MW were used). Liquid junction potentials arising from entry into the bathing solution were compensated for in the voltage-clamp mode of the patch-clamp amplifier. Once a `tight' GW seal had been formed, cell-free `inside-out' patches were obtained by retracting the electrode from the intact crypt and then passing the pipette tip through the air-water interface to avoid vesicle formation. The series capacitance was then adjusted to compensate for the fast capacitative transient observed in response to a train of voltage pulses applied to check the integrity of the G-W seal.

Currents across the patch were then recorded as outlined by Hamill et al. (1981) using a Biologic RK-300 amplifier (Biologic, Claix, France) at a gain of 50-100 mV/pA. During continuous recordings of `whole-patch' currents the patch was held at 0mV (patch potentials were referred to the extracellular side of the membrane). Alternatively the patch was held at -50mV when applying voltage pulse protocols. Junction potentials evoked following bath solution changes were determined and measurements obtained from recordings are as given in table 9.3..

9.2.3. Data acquisition and analysis.

The analysis and acquisition of data was performed as outlined in 3.2.4. with the following adaptions for excised patch recordings. During continuous current recordings the `whole-patch' current output from the patch-clamp amplifier was simultaneously monitored on a Gould chart recorder and recorded on videotape for subsequent analysis. An IBM-AT microcomputer equipped with a Labmaster interface and software written by John Dempster (VCAN, Dept. of Physiology and Pharmacology, University of Strathclyde, Scotland) was used for data acquisition and analysis of continuous current recordings. Replayed records of `whole-patch' currents were low-pass filtered at 1 kHz (-3db) using a variable 8-pole Bessel filter (Barr & Stroud) and then digitised using a PC-Lab laboratory interface. Digitised records were plotted after decimation to 4 samples per sec.

The `whole-patch' current families were monitored by applying voltage pulse protocols to the stimulus input of the Biologic RK-300 amplifier following digital-to-analogue conversion using a Lab-PC laboratory interface (National Instruments, U.S.A.). The signal from the patch-clamp amplifier was simultaneously viewed on a storage oscilloscope and recorded on videotape for subsequent analysis together with triggering pulses using an adapted pulse-code modulation encoder (Lamb, 1985). Voltage-clamp recordings were acquired at a frequency of 2-5 kHz after low-pass filtering as outlined above. Results are expressed as means ± standard errors of n observations.

9.3. RESULTS.

Patch pipettes were filled with solutions containing 145mM Na gluconate, 1.3mM CaCl₂, 0.5mM MgCl₂ and 1mM K gluconate. When the cytosolic face of the patch pipette was bathed with a solution containing 135mM Na⁺, 10mM K⁺, 148mM Cl^-, 0.5mM Mg²⁺ and 36nM Ca²⁺ reversal potentials of -59mV for K⁺, 1.8mV for Na⁺ and 95.5mV for Cl^- can be calculated (assuming a negligible permeability to gluconate^- ions) according to the Nernst equation: E_{rev X} = RT/zF.log_e([X⁺]_o /[X⁺]_i), where RT/zF = 25.67 at 25° C.

In Fig.9.1A. a continuous recording of patch current (I_p) from an excised inside-out patch originally bathed with a solution containing 0.1mM KCl and 145mM NaCl ([Ca²⁺]_i = 36nM) is shown. The recording shown in the top panel of Fig.9.1. shows the effect of increasing the concentration of K⁺ bathing the cytosolic face to 145mM by equimolar replacement of NaCl with KCl, initially with the cytosolic [Ca²⁺]_i buffered first to 1m M and then to < 10nM with EDTA. In 3 such experiments substituting 145mM KCl for NaCl mean changes in I_p of 1.25 ± 0.07 pA and 1.70 ± 0.49 pA were observed for free [Ca²⁺]_i values of 36nM and 1m M respectively. All changes in I_p evoked upon substitution of K⁺ for Na⁺ were readily reversible.

In all subsequent experiments pipette solutions were used containing 1mM KCl and 145mM Na Gluconate with the cytosolic face of the patch bathed initially with a solution of composition 10mM KCl, 135mM NaCl (free [Ca²⁺]_i buffered to 36nM) which resulted in a mean I_p of 5.28 ± 1.23 pA (n=6) when the patch was held at 0mV. Fig.9.1B shows a continuous recording of I_p in which the composition of the bathing medium was changed from 135mM NaCl, 10mM KCl to 145mM KCl, initially with free [Ca²⁺]_i buffered to 36nM, and then to 1m M. In all 7 patches in which cytosolic K⁺ was increased to 145mM KCl with cytosolic free [Ca²⁺]_i of 36nM and in all 4 patches where K⁺ was increased to 145mM with 1m M free [Ca²⁺]_i a readily reversible increase in I_p was observed (see table 9.2. for means of data ± SEM). In four paired experiments (e.g. Fig.9.1B.) no significant difference in the increase in I_p was observed between K⁺ substitution experiments in which [Ca²⁺]_i was buffered to 1m M or 36nM (1.71 ± 0.27 vs 1.68 ± 0.34 pA). Fig 9.1C. shows the effect of substituting 135mM NMGCl for 135mM NaCl in the bathing medium whilst [K⁺]_i and [Ca²⁺]_i are kept at 10mM and 36nM respectively. In all 6 patches in which Na⁺ was substituted with NMG⁺ a decrease in I_p was observed which was readily reversible. Mean changes in I_p for each ion substitution manoeuvre are given in table 9.2. The bursts of inward current in this recording may reflect channel activity gating Na⁺ currents.

Fig.9.1.
Cationic permeability of excised inside-out patches from the crypt basolateral membrane. A. Continuous recording of current across an excised patch of membrane held at 0 mV with a pipette solution containing 0.1mM KCl and 145 mM Na Gluconate (see table 1 for composition of solutions). During the periods indicated by the horizontal bars the solution bathing the intracellular face was changed from a solution of composition (mM) 145 NaCl, 0.1 KCl (with free [Ca²⁺]_i buffered to 36nM with EGTA) to a solution containing 145 KCl (equimolar replacement for NaCl) with either an unchanged (36nM) or elevated free [Ca²⁺]_i (1m M). B. Continuous recording of transmembrane current across an excised inside-out patch held at 0mV with respect to pipette containing a high Na⁺, low Cl^-, 1mM K⁺ pipette solution (see table 1). During the periods indicated by the horizontal bars the intracellular bathing solution was changed from (mM) 10 KCl, 135 NaCl (free [Ca²⁺]_i = 36nM) to 145 KCl with either an equivalent or elevated (1m M) free [Ca²⁺]_i as described above. C. Continuous current recording as in B with equimolar substitution of 135mM N-Methyl D-Glucamine for NaCl. Replacement experiments with 145 KCl as for B. Scale bars are representative for all recordings shown. Arrows indicate zero-current levels and horizontal bars the duration of solution changes.

Experiments were then performed to determine the selectivity of the cationic permeability pathway of the patch by recording the currents across the patch induced by a voltage pulse-protocol (as shown in Figs. 9.2 and 9.3). The cytosolic face of the patch was held at -50 mV, a value typical of the crypt resting membrane potential as measured by the perforated-patch technique (see section 3.3). Voltage pulses of 3 second duration were applied to the pipette in 40mV increments from -80mV to 80mV. Fig.9.2 shows the current families obtained during experiments in which the cationic composition was changed from 10mM K⁺, 135mM Na⁺ to either 145mM K⁺ or 135mM NMG⁺ and 10mM K⁺. In these experiments [Ca²⁺]_i was buffered to 36nM, a value similar to that reported from measurements recorded from unstimulated T84 cells (Devor et al., 1991). The average current at each holding potential was determined and the current-voltage (I-V) relation derived for the recording in Fig.9.2 is shown below. In this experiment the substitution of 135mM KCl for 135mM NaCl resulted in a shift in the I-V reversal potential from -12 to -26mV, associated with an increase in the magnitude of outward current at depolarised potentials and a greater outward rectification of the I-V relation.

Fig.9.2.

Cation selectivity of transmembrane currents present in excised patches from the crypt basolateral membrane. A. `Whole-patch' current families recorded from an excised inside-out patch with a Na⁺-rich, low Cl^- pipette solution containing 1mM K⁺. The intracellular face of the excised patch was held at -50mV with respect to the patch pipette. Families of voltage pulses of 3s duration were applied to the patch as indicated in the top left hand panel. Current families were recorded with an `intracellular' bathing solution containing (mM) 10 KCl and 135 NaCl (free [Ca²⁺]_i buffered to 36nM with EGTA, top right panel) and with a bathing solution changed to a Na⁺-free solution containing either 145 KCl (middle right panel) or 135 N-methyl D-glucamine Cl with the [K⁺]_i concentration kept at 10 mM (middle left panel) with no alterations in free [Ca²⁺]_i. The current, voltage and time scale bars shown are representative of all recordings shown. Arrows indicate zero-current levels.

Substitution of 135mM NaCl with 135mM NMGCl resulted in a decrease in the outward current associated with an inward rectification of the I-V relation and a shift in the I-V reversal to +8 mV. Note that the I_p at -80mV appears to be independent of the cationic composition of the bath. Table 9.3 shows the mean shifts (±SE) in reversal potentials upon cation substitution for n different patches from the control solution (10mM KCl, 135mM NaCl, 36nM [Ca²⁺]_i).

The effect of increasing intracellular free Ca²⁺ from 36nM to 1m M upon the size of the outward currents recorded with 145 mM KCl in the bathing solution was determined. Fig.9.3A shows the effect of elevating [Ca²⁺]_i upon the magnitude of the current families induced by the pulse protocol shown in Fig.9.3A. No consistent difference in either the magnitude of the reversal potential shift or the outward currents evoked was observed when the K⁺ replacement was accompanied by an increase in [Ca²⁺]_i from 36nM to 1m M. Fig.9.3B presents the averaged currents recorded as means (± SE, n=5) for all bathing solutions tested.

A time-dependent activation of outward current at more depolarised potentials was evident in 13/18 (72%) excised patches to which depolarised pulses were applied as shown in Fig.9.3A, although the time-dependent activation does not appear to be dependent upon [Ca²⁺]_i. However in 5/18 (28%) of patches linear or slowly inactivating currents at depolarised potentials were observed (see Fig.9.2). A similar voltage dependence was observed in cell attached patches, although the inability to perform ion substitution experiments prevented the characterisation of these currents (not shown).

Fig.9.3.
Calcium and voltage dependence of cationic whole-patch transmembrane currents present in excised patches from the crypt basolateral membrane. A. `Whole-patch' current families recorded from an excised inside-out patch with a Na⁺-rich, low Cl^- pipette solution containing 1mM K⁺ (table 1). The intracellular face of the excised patch was held constant at -50 mV with respect to the patch pipette. A family of voltage pulses of 3s duration were applied to the patch as indicated in the top left hand panel. Current families were recorded when the `intracellular' bathing solution contain (mM) 10 KCl, 135 NaCl (currents not shown, [Ca²⁺]_i =36nM) was changed to 145 KCl without a change in [Ca²⁺]_i (middle right panel) and then to 145 KCl with [Ca²⁺]_i buffered to 1m M. The middle left panel shows the effect of an equimolar substitution of 135mM NaCl with 135mM N-methyl D-glucamine Cl with the [K⁺]_i concentration maintained at 10 mM (middle left panel) without altering free [Ca²⁺]_i. Current, voltage and time scale bars are shown and are representative of all recordings shown. Arrows indicate zero-current levels. B. Current-voltage relations obtained from current families recorded with various concentrations of free [cation]_i and [Ca²⁺]_i. Average `whole-patch' currents determined at each holding potential from 5-6 separate experiments are presented here as means (± SE) for each of the bathing solutions tested (the number of different patches is given in brackets).

9.4. DISCUSSION.

Excised patches bathed with a solution containing 145mM Na gluconate, 1.3mM CaCl₂, 0.5mM MgCl₂ and 1mM K gluconate at the extracellular face and with a solution bathing the cytosolic face of composition 135mM Na⁺, 10mM K⁺, 148mM Cl^-, 0.5mM Mg²⁺ and 36nM Ca²⁺, reversal potentials of -59mV for K⁺, 1.8mV for Na⁺ and 95.5mV for Cl^- could be calculated if a negligible permeability to gluconate^- ions is assumed. Therefore an excised patch with such an imposed ionic gradient at 0mV will carry a net inward current if a conductance pathway is predominately anion or Na⁺-selective. Under these conditions, however, the patch consistently passed an outward current. As cytosolic [K⁺]_i was increased from 10 mM to 145 mM the outward current increased reversibly and was apparently independent of [Ca²⁺]_i. Unstimulated T84 intestinal cells have been demonstrated to have a resting [Ca²⁺]_i of between 30 and 40 nM and a value that may approach 500 nM in cells stimulated by 100m M carbachol (Devor et al., 1991). Thus a [Ca²⁺]_i of 1m M may exceed the [Ca²⁺]_i that occurs following carbachol stimulation in crypt enterocytes, strengthening the contention that this cationic permeability is Ca²⁺-independent, although this does not preclude modulation by other intracellular mediators such as calmodulin which may be lost upon patch excision.

The increase in outward current and the negative shift in the reversal potential associated with substitution of Na⁺ with K⁺ is consistent with the presence of a permeability pathway that is selective for K⁺ over Na⁺ ions. However the large decrease in outward current and the positive shift in the reversal potential that occurs upon substitution of Na⁺ with NMG⁺ suggests a substantial permeability to Na⁺ ions, as the [K⁺]_i was not altered. The mean reversal potential obtained with cytosolic concentrations of 10mM KCl and 135mM NaCl was -17 mV, from which value a P_K/P_Na of 18:1 could be calculated using the Goldman-Hodgkin-Katz (GHK) equation in the form;

E_REV = RT/zF. ln ((P_Na.[Na⁺]_o + P_K.[K⁺]_o) / (P_Na.[Na⁺]_i + P_K.[K⁺]_i))

assuming that no leak permeability component was present. This assumption is unlikely to be valid and therefore such a value is probably an underestimate of the P_K/P_Na ratio. Following an increase in cytosolic KCl from 10 to 145 mM the mean reversal shifted to -27 mV, from which a P_K/P_Na of 2.9 was calculated, considerably lower than the value obtained with 10 K⁺ in the bathing medium. Assuming a P_K/P_Na of 2.9, a P_Na/P_NMG of 2.44 and a P_K/P_NMG of 9.1 may be calculated from the mean reversal potentials shown in Fig. 9.3B. The discrepancy between the apparent permeability ratios obtained with 145mM KCl and 10 mM KCl could not be accounted for by a junction potential or by a deterioration in the integrity of the seal, as the changes in whole-patch current and reversal potential were completely reversible. One proposal to explain this phenomenon is that the selectivity of this pathway may be regulated in some manner by the cytosolic Na⁺ and/or K⁺ activities, with the selectivity of this pathway to K⁺ appearing to decrease as the intracellular [K⁺]_i is increased and the intracellular [Na⁺]_i is decreased.

No clear open levels could be discerned, even at depolarised holding potentials, irrespective of the cytosolic [Ca²⁺]_i. Two lines of argument suggest that a single channel conductance is present, rather than a selective `leak' pathway or an electrogenic ion transporter. Firstly, outward current `noise' increases at depolarised potentials, consistent with an increase in the electrochemical gradient for K⁺ movement into the pipette. Secondly there is a time-dependent activation of outward current at more depolarised potentials that is not apparently due to the deterioration of the seal resistance. If this cation selective pathway is in fact a regulated channel protein then its conductance must be less than 1pS. However, the possibility that some form of electrogenic transport protein is present cannot be excluded. The phenomenon of time-dependent activation of outward current at depolarised holding potentials was also observed in the cell-attached configuration, although the selectivity of these currents could not be determined, suggesting that the depolarisation-induced activation of outward current was not due to the loss of an intracellular component or mechanical disruption associated with patch excision. The variation in the time-dependence in outward currents between patches could not be explained.

The outwardly-rectifying and time-dependent K⁺ currents recorded from excised patches obtained from the crypt basolateral membrane are consistent with the whole-cell K⁺ currents described in chapter 3. The outward-rectification of the membrane patch currents and those obtained from perforated-patch whole-cell recordings may be explained in part by Goldman rectification due to the asymmetrical K⁺ concentration across the membrane. However the outward rectification of these currents was not as pronounced as those recorded in the whole-cell configuration in the unstimulated crypt, although this may be explained by the comparatively greater leak component of the excised patch currents, and the possible presence of an another outwardly rectifying conductance in the apical membrane (Sepulveda et al., 1991) and a depolarisation-activated basolateral K⁺ conductance in the crypt enterocyte. The contention that this cationic permeability may be responsible for determining the resting membrane potential in small intestinal crypts is consistent with the basolateral localisation of this permeability, its apparent Ca²⁺ independence and K⁺ selectivity. The permeability ratio of K⁺ to Na⁺ ions appears to be higher at low [K⁺]_i, with a P_K/P_Na of >18:1 which falls to >3:1 as intracellular K⁺ increases and [Na⁺]_i is eliminated. This may represent a feedback mechanism, whereby Na⁺ influx into the cell may reduce the relative permeability of the membrane to Na⁺ ions, thus repolarising the membrane potential. If such a mechanism was coupled to Na⁺-K⁺ ATPase activity it may play a role in regulating the intracellular K⁺ activity.

Studies of the basolateral membrane conductance present in apically permeabilised epithelial monolayers of the A6 amphibian cell line have revealed that under basal conditions the basolateral K⁺ conductance is inwardly-rectifying and quinine-insensitive, and that this conductance becomes outwardly-rectifying and quinine-sensitive after the cell is swollen by increasing the apical Cl^- concentration (Broillet & Horisberger, 1991). Similarly elevating the `mucosal' Cl^- concentration in apically-permeabilised HT-29 cell monolayers has also been reported to activate a quinine-sensitive basolateral K⁺ conductance (Illek et al., 1992). Neither study addressed the relative permeability of this conductance to Na⁺ ions. These observations raise the possibility that the high pipette Cl^- concentrations used in crypt perforated-patch experiments may swell the cell, thus activating the outwardly-rectifying whole-cell K⁺ currents observed (Rae & Fernandez, 1991). However the outwardly-rectifying K⁺ currents are also present in excised patches obtained without prior exposure of the crypt to hypotonic media. Furthermore Ba²⁺ and quinine both evoke depolarisations of membrane potential in unstimulated crypts, as well as blocking regulatory volume decrease following hypotonic swelling. It is therefore proposed that this basolateral K⁺-selective cationic permeability plays a major role in determining the resting membrane potential in the crypt enterocyte.

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Table 9.1. Composition of solutions used in ion substitution experiments.

	Hanks	0.1mM K⁺ pipette	1mM K⁺ pipette	0.1mM K⁺ bath	10mM K⁺ 135Na⁺	145 KCl low Ca²⁺	145 KCl high Ca²⁺	10mM K⁺ Na⁺ free
NaCl	140	-	-	145	135	-	-	-
KCl	5	-	-	0.1	10	145	145	10
CaCl₂	1.3	1.3	1.3	0.1	0.1	0.1	1.91	0.1
EGTA	-	-	-	0.25	0.25	0.25	2	0.25
MgCl₂	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
HEPES	10	10	10	10	10	10	10	10
Na Gluconate	-	145	145	-	-	-	-	-
K Gluconate	-	0.1	1	-	-	-	-	-
K₂HPO₄	0.36	-	-	-	-	-	-	-
KH₂PO₄	0.44	-	-	-	-	-	-	-
NaHCO₃	4.2	-	-	-	-	-	-	-
N-methyl-D-glucamine Cl	-	-	-	-	-	-	-	135

All concentrations given in mM and solutions titrated to pH 7.2 with Tris.

Change in composition of intracellular solution (concentrations are given in mM, {[Ca²⁺]_i})	Mean change in I_out (pA)	± standard error	(n)
10 KCl, 135 NaCl {36nM} ® 145 KCl {36nM}	2.67	0.54	7
10 KCl, 135 NaCl {36nM} ® 145 KCl {1m M}	1.71	0.26	4
10 KCl, 135 NaCl {36nM} ® 10 KCl, 135 NMGCl {36nM}	-5.47	1.13	6

Table 9.2.

Mean changes in outward current (I_out) given in pA (± SE) for n different excised inside-out basolateral patches obtained with pipettes of resistance 8-10 MÛ containing a solution of composition (mM) {1 KGluconate, 145 NaGluconate, 1 CaCl₂, 0.5 MgCl₂, 10 HEPES, pH 7.2 with Tris base}. Changes in the values of outward current are presented as means of paired values (Change in I_out = I_out {test solution} - I_out {10 KCl, 135 NaCl, 36nM [Ca²⁺]_i control}).

Change in composition of intracellular bathing solution (concentrations are given in mM, {[Ca²⁺]_i})	Mean shift in reversal potential from 10KCl, 135NaCl control (mV)	± standard error	Junction potential (mV)	(n)
10 KCl, 135 NaCl, {36nM} ® 145 KCl, {36nM}	-10.0	1.4	-0.1	4
10 KCl, 135 NaCl, {36nM} ® 145 KCl, {1m M}	-10.8	0.95	-0.1	4
10 KCl, 135 NaCl, {36nM} ® 10 KCl, 135 NMGCl, {36nM}	+31.0	4.2	-1.5	4

Table 9.3.

Mean changes in the extrapolated zero-current reversal potentials upon the solution changes indicated, given in mV (± SE), for n different excised inside-out patches with 8-10 MÛ pipettes filled with solution of composition (mM) {1 KGluconate, 145 NaGluconate, 1 CaCl₂, 0.5 MgCl₂, 10 HEPES, pH 7.2 with Tris base}. Shifts in reversal potentials are presented as means of paired values of the test solution compared to the 10 KCl, 135 NaCl control solution (see also Fig.3.B.). Junction potentials measured in the voltage-clamp mode are given.