Doi:10.1080/10647440400003899

Molecular Membrane Biology, September Á/October 2004, 21, 307 Á/313 pH modulation of large conductance potassium channel from adrenalchromaffin granules channel gene CLCN7 leads to a severe osteopetrotic phenotype because osteoclasts fail to resorb bone and they cannot acidify the lacuna [6]. Mitochondrial potassiumchannel has been suggested as a trigger and effectormyocardial ischemic preconditioning [7].
$ Department of Biophysics, Agriculture University SGGW, Ion channels have also been reported in the membrane of 159 Nowoursynowska St., 02-776 Warsaw, Poland chromaffin granules from adrenal medulla [8 Á/12]. Thechromaffin granules are involved in catecholamine synthesis % Laboratory of Intracellular Ion Channels, Nencki Institute of and traffic, both within and outside the cell [13]. The uptake of Experimental Biology, Polish Academy of Sciences, 3 hormones, driven by pH gradient (DpH), from the cytosol into chromaffin granules is catalyzed by a catecholamine carrier § Laboratory of Protein Chemistry, Institute of Bioorganic [14]. Secretion of hormones occurs as a result of the fusion of Chemistry, Belarus National Academy of Sciences, chromaffin granule vesicles with the plasma membrane of The chromaffin granule ion channels have been investi- gated after fusion of granule membranes with bilayermembrane (BLM), followed by single channel recordings.
We report here that large conductance K selective channel inadrenal chromaffin granules is controlled by pH. We measured Several different cation selective channels were described electrogenic influx of 86Rb into chromaffin granules prepared after incorporation of intact chromaffin granules, but only two from bovine adrenal gland medulla. The 86Rb influx was types of highly selective K' channels could be reconstituted inhibited by acidic pH. Purified chromaffin granule membranes from preparation of chromaffin granule ‘‘ghosts’’ [11,12]. A were also fused with planar lipid bilayer. A potassium channelwith conductance of 4329 K' selective, large conductance ( Â/160 pS in symmetrical observed after reconstitution into lipid bilayer. The channel 400 mM KCl) channel was described by Arispe et al . [11]. It activity was unaffected by charybdotoxin, a blocker of the was insensitive to charybdotoxin, a blocker of the Ca2'- Ca2-activated K channel of large conductance. It was activated K' channel of large conductance [11]. The channel observed that acidification to pH 6.4 cis side of the membranelowered the channel open probability and single channel activity was also unaffected by Ca2' and potential across conductance. Whereas only weak influence on the single the bilayer [11]. It was also reported that the chromaffin channel current amplitude and open probability were observed granule K' channel was controlled by both inhibitory and upon lowering of the pH at the trans side. We conclude that a stimulatory heterotrimeric GTP-binding proteins [15]. A pH-sensitive large conductance potassium channel operates inthe chromaffin granule membrane.
similar channel, highly selective for potassium, but with adifferent conductance ( Â/400 pS in symmetric 450 mM KCl), Keywords: intracellular chromaffin granule potassium channel, was described by Ashley et al . [12]. The channel was adrenal chromaffin granules, bilayer lipid membranes.
insensitive to both Ca2' and charybdotoxin, and wasblocked by TEA'.
A key problem concerning single channel recordings of BLM, blacklipid membrane technique; I, single-channel current chromaffin granules in planar bilayer membranes is the purity amplitude; U, potential; Urev, reversal potential; to, mean lifetime Á/ of the applied membrane preparation. Therefore, to study open time; tc, mean lifetime Á/ closed time; Popen, open-probability; chromaffin granule potassium transport we applied both single channel recordings and flux measurements using86 Rb , a K' analog, as described by others [16,17].
Previously we have successfully used this approach to show that electrogenic K' transport in chromaffin granules Potassium and chloride selective channels exist in mem- is blocked by sulfhydryl reagents [18], various potassium branes of organelles such as mitochondria, sarco/endoplas- channel blockers [19] and by ATP [20]. Transport measure- mic reticulum, endosomes, synaptic vesicles and secretory ments were performed under such conditions that only an granules [1 Á/4]. They are involved in intracellular ion traffic electrogenic influx of 86Rb' into chromaffin granules was and play a vital role in cellular function. For example, loss of measured. This simple and convenient flux assay, combined endosome-associated chloride channel, in Dent’s disease, with marker enzyme estimations, forms a valuable method strongly inhibits endocytosis of low molecular weight proteins for measuring K' channel activity of chromaffin membrane in kidney proximal tubular cells [5]. Mutations in the Cl( vesicles. This kind of approach together with single channelmeasurements after reconstitution into planar lipid bilayerallows us to study new properties of the chromaffin granules *To whom correspondence should be addressed.
e-mail: adam@nencki.gov.pl ISSN 0968-7688 print/ISSN 1464-5203 online # 2004 Taylor & Francis Ltd In this paper we report that the large conductance potassium channel present in chromaffin granule mem- [K+] > > [K+]
branes is regulated by pH. The potassium channel was investigated both by 86Rb' ion flux measurements and single channel recordings after reconstitution into blacklipidmembrane (BLM). Both techniques showed that the chro-maffin granule potassium channel is inhibited by the acidic pH, probably from the intragranular side.
[K+] = [K+]
[% of total radioactivity]
Regulation of 86Rb' uptake into chromaffin granules by pH The principle of the applied flux assay was originally Time [min]
described by others [16,17]. In brief, we prepared chromaffingranule vesicles containing an inner concentration of 100 mM KCl. Shortly before the assay external K' wasreplaced with Tris'. As a result of a K' gradient, an electrical diffusion potential was established in vesicles containing active K' channels. The addition of isotope, a K' analog, to the external solution, led to the uptake of Rb' due to its equilibration with the membrane potential, but not affecting the level of the potential itself. It is important to note that 86Rb' accumulation occurs selec-tively into the vesicles containing open K' channels thus [fold of stimulation
enhancing the sensitivity of the transport measurements.
Figure 1 (a) presents the time course of 86Rb' uptake into chromaffin granule vesicles (expressed as the percentage of total radioactivity present in the sample). Addition of 30 mM KCl, which caused depolarization of the diffusion potential,promoted a rapid efflux of 86Rb' from the vesicles. In the 86Rb' uptake into chromaffin granules and its regulation absence of a K' gradient (no diffusion potential was created) by pH. (a) Time course of 86Rb' uptake into chromaffin granules.
After addition of 86RbCl, accumulation of radioactivity was measured accumulation of 86Rb' was low (Figure 1 (a)). This result as described in ‘‘Materials and Methods’’ (j). At the time indicated suggests that the K' transport pathway operates by an by an arrow, 30 mM KCl was added to the reaction mixture (m).
electrogenic rather than electroneutral mechanism.
Accumulation of radioactivity without removal of external potassium Figure 1 (b) demonstrates the effect of the pH of the is also shown ('). Values are means9/S.D. for triplicate determina- incubation medium on 86Rb' uptake into chromaffin granule tion. Measurements were performed at 208C. (b) Effect of pH on 86Rb' uptake into chromaffin granules. The uptake was measured vesicles. A large inhibition of 86Rb' uptake was observed in at different pH values as described under Materials and Methods.
a medium of pH below 7.0. In order to verify whether the The 86Rb' uptake at pH 7.0 was taken as unity. Values are means9/ observed effect was specific to chromaffin granule mem- branes, a similar experiment was performed with beef heartsubmitochondrial particles (SMP), known to have only aslightly pH-dependent electrogenic potassium transport. In KCl after addition of the vesicles into the trans side. Anion fact, no inhibition of 86Rb' transport was observed in beef selective channels were observed in only Â/2% of all experiments. The potassium channels were usually similarin amplitude and gating behavior from experiment to experi- Reconstitution of chromaffin granule membranes into planar ment. Examples of single-channel recordings of the potas- sium channel are illustrated in Figure 2. We calculated thecurrent/voltage (I/V) relation from the mean amplitude of the Figure 2 shows current changes upon reconstitution of channels currents at different potentials as 3609/7 pS (n 0/ chromaffin granules into a planar lipid bilayer. The quality 22) in a KCl gradient (450/150 mM KCl) (Figure 2 (a)) and of the lipid bilayers was checked at 0 mV and 9/50 mV before addition of chromaffin granule membranes suspension and 9/9 pS (n 0/7) in symmetric 450 mM KCl (data not showed no channel-like activity. Electrically silent mem- shown), both at pH 7.0. Channel had an ohmic behavior between '/70 and (/70 mV, both in symmetrical solution of 10 pS. We routinely observed a positive current at 0 mV, (open squares) and in presence of 450/150 mM KCl ionic gradient (closed squares) (Figure 2 (b)). The selectivity of the /50 minutes after addition of chromaffin granule ‘‘ghosts’’ to the trans -bilayer chamber. These could be identified as observed fluxes through this channel was investigated with due to a cation selective channel by the direction of current asymmetric KCl solutions. The obtained reversal potential flow in the presence of an ion gradient 450 mM KCl/150 mM was Urev 0/(/289/2 mV, indicating that the channel is highly Calculated probability of opening for 50 mV in symmetrical450 mM KCl was Popen0/0.519/0.15 whereas for (/50 mVwas only Popen0/0.119/0.07. Such a result clearly indicatedthat potassium channel with large conductance is voltagedependent. We also performed gating analysis of singlechannel recording at 50 mV and (/50 mV in symmetrical450 mM KCl, pH 7.0. Single current amplitudes at 9/50 mVobtained from histograms had the same values ( Â/229/1 pA)whereas strong differences were observed in the closed- andopen-time distributions. The calculated open-times for thelarge conductance potassium channel investigated are:to 0/9.749/0.37 ms at 50 mV but only to 0/3.859/0.31 ms at (/50 mV. The effect was more evident for the closed-times: tc0/10.189/0.38 ms at 50 mV and tc0/31.989/0.92 ms at (/50 mV, suggesting that the channel is voltage dependent.
The minimum number of open and closed states entered byKCG channel can be estimated determining the number ofexponential components necessary to fit the observe open-and closed- time distribution [22]. Open- and closed-timehistograms of the KCG channel in 90% of experiments clearlyrequired only one time constant to fit the points. In remaining10% of experiments second lifetimes-lower than 3 ms weregenerated but they determined not significant (only Â/1%)per cent of all. Such a result points that investigated channelhas only one population of open- and closed times.
Charybdotoxin has no effect on the potassium channel activity upon application to either the cis or trans side (datanot shown).
Figure 3 shows KCG channel activity at holding potential 40 mV in control experimental conditions and after additionTEA' to the trans and cis sides. Experiments wereperformed in ion gradient concentration of 450 mM KCl/150 mM KCl, pH 7.0. 15 mM TEA' applied to the cis sideclearly blocked the channel. The same TEA' concentration Reconstitution of chromaffin granule large conductance potassium channel into planar lipid bilayer. (a) Single channelrecordings at different holding potentials in asymmetric ionic condi-tions (450/150 mM KCl, pH 7.0 cis /trans ). Closed levels are markedas c. Recordings were low Á/pass filtered at 200 Hz. (b) Current/voltage relation for single channel recordings in symmetric andasymmetric ionic conditions. The stright line was fitted to experi-mental data; in symmetric 450 mM KCl (I), and in asymmetricconditions 450/150 mM KCl cis/trans (j). The observed single-channel conductances are 4329/9 pS for symmetric and 3609/7 pSfor asymmetric ionic conditions.
Effect of TEA' on single channel activity. Channel selective for cations (the K' Nernstian potential for this ionic recordings at holding potential of 40 mV under control conditions in asymmetric 450 mM KCl/150 mM KCl, pH 7.0 cis /trans ) and with 15 mM TEA' applied to the trans and cis sides as indicated. Closed Probability of opening was lower for single channel levels are marked as c. Recordings were low Á/pass filtered at activity when negative potential was applied to the bilayer.
placed into the trans -bilayer chamber results in current increased to tc0/8.89/0.3 ms but after lowering it in the cis amplitude reduction at 20% without changing open prob- side it increased to tc0/30.59/2.9 ms.
ability and gating. These findings pointed on the fact that Lowering of the pH in the cis side resulted in diminishing of TEA'-sensitive channel was located in resealed ‘‘ghost’’ both open probability and the amplitude of opening. Only membranes and its’ sensitivity is higher from the cis side weakinfluence on single channel current amplitude and open named as an intragranular side. These results are consistent probability were observed when pH 6.4 was applied from the with one presented by Ashley et al . (1994).
We performed gating analysis of single channel recording Figure 5 shows the effect of pH on the open probability of in control and after addition 15 mM TEA' to the trans -bilayer the large conductance potassium channel. The significant chamber. As occurred, the calculated open- and closed- difference in probability of opening at the level PopenB/0.05 times stayed unchanged. Open- and closed- times for the (test T-Student) was observed only after lowering pH from large conductance potassium channel at 40 mV in gradient the cis side when compared with open probability at pH 7.0 at both the cis and trans sides as a control. Upon changing o 0/14.339/0.24 ms and tc 0/13.789/1.78 ms in the pH from 7.0 to 6.4 from the cis side the open probability o 0/14.439/0.26 ms and tc 0/9.329/1.74 ms after 15 mM TEA' addition to the trans -bilayer chamber.
decreased from Popen0/0.619/0.21 to Popen0/0.139/0.11.
Regulation of chromaffin granule large conductancepotassium channel by pH Protons influence the behavior a variety of ion channelsincluding potassium channels [23 Á/26]. Unitary conductance Figure 4 shows single channel recordings at holding potential of BK channels is also reduced by protons [27,28]. The of 30 mV after reconstitution of chromaffin granule mem- magnitude of K' current probably is controlled by glutamate branes into lipid bilayers in the gradient of 450/150 mM KCl residues present there [25,29]. Recently, it was shown that (cis /trans ) and kinetic analysis performed for activity of KCG low pH reduced the unitary current in a voltage dependent channel in such experimental conditions. Figure 4 (a) shows manner-increasing with the membrane depolarization [26].
channel recordings after lowering pH from 7.0 to 6.4 only at Heterologously expressed hSlo1 BK channels were also pH- the cis side and again change to the pH 7.0. Open- and closed- time histograms for various pH values of the The value of the presented results and interpretation experimental solution in the trans and cis sides are of the data are due to application of two different transport presented in Figure 4 (b). Considerable differences in the methods reflecting K' conductance in chromaffin granule lifetimes were observed at pH 6.4 at the cis side. In the membranes. The radioactive flux assay, applied in this control pH 7.0 in both compartments the closed lifetime was report, was successfully used to show amiloride-block- tc0/7.79/0.3 ms. After lowering the pH in the trans side it able Na' channels in toad bladder microsomes, and Regulation of chromaffin granule large conductance potassium channel by pH. (a) Single channel recording at a holding potential of 30 mV from ‘‘ghost’’ membranes incorporated into bilayer in the presence of gradient 450/150 mM (cis /trans ) KCl and at pH 7.0 or 6.4. A changein channel kinetics at pH 6.4 at the cis side. The pH at the trans side was always 7.0. The closed levels, corresponding to the current through thelipid bilayer, are indicated with c. Recordings were low Á/pass filtered at 200 Hz. (b) Gating analysis of large conductance potassium channelrecordings at different pH. Open- and closed- time analysis of the channel recordings at various pH values at the trans and cis sides. ThepH values at the trans and cis sides are marked below the diagram. The significant difference in the time constant values are marked by asterisk.
Mean open- (to) and closed (tc) lifetimes are indicated in ms. Data are means9/SD (n 0/7).
both insensitive to ChTX and Ca2'. Our results indicate thatthe channel activity observed in the present paper wassimilar to one described by Ashley et al . (1994). Similar to86Rb' flux experiments we observed a strong inhibition ofthe K' channel by low pH. Interestingly, this strong effectwas observed only from the cis side. The inhibition of the K'channel by lowering pH was observed from the same side asTEA' inhibition. Previously it was shown that a chromaffingranule K' channel is blocked by TEA' from the intragra-nular side [12]. This suggests that the effect of pH is alsofrom the intragranular side. The findings of the present studymay be important for our understanding of the physiologicalrole of potassium conductance in chromaffin granules.
The chromaffin granule membrane contains a vacuolar- type (V-type) H'-ATPase which generates an electrochemi-cal proton gradient, acidifying the granule interior [32]. Thepotassium channel may play an important physiological role Effect of pH on the open probability of chromaffin granule large conductance potassium channel. Changes of the pH values at by compensating for the electric charge transfer produced by the trans and cis sides are marked below the diagram. Columns and the V-ATPase [12]. This would enable formation of a error bars indicate means9/SD (n0/7). The significant difference in membrane potential and DpH, sufficient to drive catechola- the probability of opening is marked by asterisk. PopenB/0.05 (test T- mine uptake into the chromaffin granules. This hypothesis is Student) compared with open probability at pH 7.0 at both the cisand trans sides.
also supported by experiments on the effects of intra-granular cation composition on ATP-dependent acidification veratridine-activated tetrodotoxin-blockable Na' channels in of chromaffin granules [12]. In fact, a much higher DpH was rat brain synaptic membranes [16,17]. The 86Rb' flux observed with K' inside than with TEA [12]. Our present method, was previously used to study K' transport in observation on the pH-dependence of K' transport points to the fact that low pH should blockthis ‘‘charge compensation’’ ‘‘concentrative uptake’’, was also applied to measure the mechanism. Blockage of K' channels by low pH would block activity of Cl( channels from Torpedo californica elektroplax further acidification of chromaffin granules. This channel be plasma membrane [31]. The principle of the assay is as involved in protective mechanism to prevent over-acidifica- follows. Chromaffin granule vesicles are prepared to contain tion of the granular lumen. Regulation of chromaffin granuleK' transport by pH could be also important during granule a high concentration of KCl. Shortly before the assay, the swelling, playing a role, e.g., in the fusion of the granule with external potassium is replaced by the relatively impermeant the plasma membrane. In fact, chromaffin granule swelling Tris' ion. As a consequences of the potassium gradient has been observed to be regulated by internal pH [33].
created, an electrical diffusion potential is set up, the In conclusion, the results of our investigation support the magnitude of which is determined by the permeabilities of concept of the existence of an electrogenic, pH-regulated K' K', Cl( and Tris' through the membrane. Only in the transport system in chromaffin granules. Our results support vesicles containing active potassium channels is the K' the previous observations that K' channels are present in permeability likely to be much greater than the Cl( and Tris' chromaffin granule membranes [11,12]. Such a system permeabilities, and hence in these vesicles only a potassium appears to fulfill an important physiological role in the diffusion potential, interior negative, is formed. An isotope creation of transmembrane potential (DC) and a proton that permeates through the channel (in our case 86Rb') concentration gradient (DpH) across the granule membrane when added to the exterior solution will tend to equilibrate with the membrane potential and thus will accumulate in thevesicles that have formed a membrane potential (DC).
We observed that 86Rb' transport into chromaffin gran- ules is strongly pH-dependent. A large inhibition of 86Rb' uptake in a medium of pH below 7.0 was observed. Thisresult strongly indicated an effect of pH on the K' channel Subcellular fractionation of adrenal glands present in chromaffin granules. However a contribution of Bovine adrenal medullas were fractionated essentially as previously 86Rb'/K' electroneutral exchange or changes of chloride described by [21]. Purification of chromaffin granules and the purity conductance in the observed effect cannot be excluded.
of membrane preparations were confirmed by a marker enzymeestimation as previously described [20].
Hence, further studies on single channel activities of thechromaffin granule K' channels and their regulation by pHwere performed.
Reconstitution of chromaffin granule membranes revealed The isotope flux through ion-conducting pathways was performed the presence of a potassium channel with a conductance of essentially as described by Garty et al . [16,17]. Application of 86Rb' Â/430 pS in symmetric 450 mM KCl. Previously, two different flux for K' transport measurements in chromaffin granules experi- K' channels were described in chromaffin granules [11,12], ments was described previously [20].
Granule membrane marker cytochromu b561 activity was also measured by the difference between dithionite-reduced and oxidizedstates at 429 nm [34].
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Received 4 May 2004; and in revised form 24 June 2004.

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