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Functional Effects of Auxiliary ß4-Subunit on Rat Large-Conductance Ca²⁺-Activated K⁺ Channel

Laboratory of Molecular Neurobiology, Department of Life Science, Kwangju Institute of Science and Technology, Gwangju, 500-712, Korea

Correspondence: Address reprint requests to Chul-Seung Park, PhD, Laboratory of Molecular Neurobiology, Dept. of Life Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Gwangju, 500-712, Korea. Tel.: 82-62-970-2489; Fax: 82-62-970-2484; E-mail: cspark{at}kjist.ac.kr.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Large-conductance calcium-activated potassium (BK_Ca) channels are composed of the pore-forming -subunit and the auxiliary ß-subunits. The ß4-subunit is dominantly expressed in the mammalian central nervous system. To understand the physiological roles of the ß4-subunit on the BK_Ca channel -subunit (Slo), we isolated a full-length complementary DNA of rat ß4-subunit (rß4), expressed heterolgously in Xenopus oocytes, and investigated the detailed functional effects using electrophysiological means. When expressed together with rat Slo (rSlo), rß4 profoundly altered the gating characteristics of the Slo channel. At a given concentration of intracellular Ca²⁺, rSlo/rß4 channels were more sensitive to transmembrane voltage changes. The activation and deactivation rates of macroscopic currents were decreased in a Ca²⁺-dependent manner. The channel activation by Ca²⁺ became more cooperative by the coexpression of rß4. Single-channel recordings showed that the increased Hill coefficient for Ca²⁺ was due to the changes in the open probability of the rSlo/rß4 channel. Single BK_Ca channels composed of rSlo and rß4 also exhibited slower kinetics for steady-state gating compared with rSlo channels. Dwell times of both open and closed events were significantly increased. Because BK_Ca channels are known to modulate neuroexcitability and the expression of the ß4-subunit is highly concentrated in certain subregions of brain, the electrophysiological properties of individual neurons should be affected profoundly by the expression of this second subunit.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The large-conductance calcium-activated potassium channels (BK_Ca or Maxi-K channels) are a family of potassium-selective ion channels activated in response to the increased intracellular calcium concentration and the transmembrane voltages (for review, Sah, 1996; Toro et al., 1998; Weiger et al., 2002). BK_Ca channels are found in a variety of both electrically excitable and nonexcitable cells (Jan and Jan, 1997). Their activities play a key role in neuronal signaling, because they are involved in regulation of transmitter release and repolarization of action potentials (Robitaille and Charlton, 1992; Robitaille et al., 1993; Bielefeldt and Jackson, 1994; Kaczorowski et al., 1996). A single gene, slowpoke (slo), was initially identified to encode BK_Ca channels in Drosophila and the molecular cloning of mammalian ortholog (Slo) were soon followed (Atkinson et al., 1991; Adelman et al., 1992; Butler et al., 1993; Tseng-Crank et al., 1994; Rosenblatt et al., 1997; Ha et al., 2000). The Slo protein alone can form functional potassium channels of Ca²⁺ and voltage dependence, and a wide range of functionally different BK_Ca channels can be expressed by the extensive splicing variations of Slo RNA as well as the modulation of channel activity through intracellular signaling mechanism (Shipston, 2001; Tian et al., 2001). In addition to the Slo protein (-subunit), the second subunits (ß-subunits) of BK_Ca channels were identified in higher mammals. The coexpression of the auxiliary ß-subunits can change the biophysical and pharmacological properties of the channel composed of only -subunits (McManus et al., 1995; Valverde et al., 1999; Wallner et al., 1999; Xia et al., 1999; Qian et al., 2002).

Four distinct ß-subunits of the BK_Ca channel were identified and their genes were cloned from various sources. Although the coexpression of the ß1-subunit with the BK_Ca -subunit was found to increase Ca²⁺ sensitivity, slow gating kinetics, and alter pharmacological properties (McManus et al., 1995; Knaus et al., 1994; Dworetzky et al., 1996), the ß2-subunit resulted in rapid and complete inactivation of BK_Ca currents (Wallner et al., 1999; Xia et al., 1999; Uebele et al., 2000). The ß3-subunit also inactivated the BK_Ca channel currents rapidly but incompletely. This subunit increased the rate of current activation at moderate Ca²⁺ concentration and induced inward rectification of the current-voltage relationship (Uebele et al., 2000; Brenner et al., 2000; Xia et al., 2000). The ß4-subunit was most recently identified and its complementary DNAs (cDNAs) were cloned from human and mouse. The ß4-subunit of BK_Ca channels was shown to express exclusively in neuronal tissues including cerebral cortex, cerebellum, hippocampus, pons, medulla, thalamus, spinal cord, and so on. Although the ß4-subunit is dominantly expressed in brain, the coexpression of this subunit affects the properties of macroscopic BK_Ca channel currents only in a modest manner (Brenner et al., 2000; Behrens et al., 2000; Weiger et al., 2000). It was noticed that the coexpression of human ß4-subunit slowed down the activation and the deactivation rate of human Slo expressed in heterologous systems. Although the ß4-subunit certainly affected the Ca²⁺-dependent activation of macroscopic human Slo currents, the effect of the ß4-subunit was somewhat inconsistent among the previous reports (Brenner et al., 2000; Behrens et al., 2000; Weiger et al., 2000). Thus, to understand its physiological roles in neuronal cells, it is important to understand the detailed functional effects of the ß4-subunit.

In this study, we investigated the electrophysiological effects of rat ß4-subunit at the level of both macroscopic and single-channel currents in various concentrations of intracellular Ca²⁺. We first isolated the full-length cDNA of the ß4-subunit from the rat brain library and expressed in Xenopus oocytes together with rSlo. This study shows that the ß4-subunit increases the sensitivity for voltage and the cooperativity for intracellular Ca²⁺. The ß4-subunit markedly alters the gating behavior of BK_Ca channels. The activation and deactivation rates are significantly lowered by the coexpression of the ß4-subunit, as shown in a previous study. However, the effects were highly dependent upon the intracellular concentration of Ca²⁺. In addition, the single-channel recordings revealed that the steady-state gating kinetics rSlo/rß4 channels were much slower than that of rSlo. Therefore, the ß4-subunit affects the electrophysiological activity of the BK_Ca channel -subunit and thus may modulate the excitability of the neuron accordingly.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Isolation of cDNA for rat BK_Ca channel ß4-subunit by library screening
A cDNA library of rat total brain in gt11 (Clontech, Palo Alto, CA) was screened by plaque hybridization method to isolate the full-length cDNA of the BK_Ca channel ß4-subunit (rß4). The entire open reading-frame (ORF) region of human ß4 cDNA (KCNMB4 or hß4) was used as a hybridization probe for initial screening. The hß4 cDNA was amplified by polymerase chain reaction (PCR), gel purified, and then labeled with ³²P using random-primer extension (Amersham Pharmacia, Piscataway, NJ). Positive clones of the first-round screening were further purified up to four consecutive rounds. The DNA inserts were isolated and subcloned into a plasmid, pBluescript II (Stratagene, La Jolla, CA). DNA sequences of the inserts were determined more than three times in both directions using an automatic DNA sequencer (Perkin-Elmer, Shelton, CT).

Functional expression of rß4 and rat BK_Ca channel -subunit in Xenopus oocytes
The rß4 and the rat BK_Ca channel -subunit (rSlo) cDNAs were subcloned into pGH vector for expression in Xenopus oocytes. The sequence information of rSlo used in this study can be obtained from GenBank accession number AF135265 (Ha et al., 2000). This rSlo clone is expressed highly in rat brain and contains the insertions of 4, 0, 0, and 27 amino acid residues at splicing sites 1–4, respectively, when compared with that of human Slo (GenBank accession number U11717) (Tseng-Crank et al., 1994). The pGH expression vector contains the 5'- and 3'-untranslated regions of Xenopus ß-globin gene and is known to enhance the protein expression of certain mammalian messages in Xenopus oocytes (Liman et al., 1992). Complementary RNA of each construct was prepared in vitro as described in previous studies (Ha et al., 2000; Soh and Park, 2001). Plasmid DNA was purified (Qiagen midi-prep columns, Qiagen, Valencia, CA) and digested with a restriction enzyme, NotI. Complementary RNA (cRNA) was synthesized from linearized plasmid DNA using T7 RNA polymerase in the presence of a cap analog, m7G(5')ppp(5')G, and nucleotide triphosphates. Oocytes of stages V and VI were surgically removed from the ovarian lobes of anesthetized female Xenopus laevis (XenopusOne, Dexter, MI) and transferred into Ca²⁺-free OR medium (86 mM NaCl, 1.5 mM KCl, 2 mM MgCl₂, 10 mM HEPES, 50 µg/ml gentamycin (pH 7.6)). The follicular cell layer was removed by incubating oocytes in Ca²⁺-free OR medium containing 3 mg/ml collagenases (Worthington Biochemical, Lakewood, NJ) for 2 h. The oocytes were then washed extensively with and kept in ND-96 medium (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, 50 µg/ml gentamicin (pH 7.6)) at 18°C. Each oocyte was injected with 50 nl of cRNA containing 1 ng for single-channel recordings and 50 ng for macroscopic current recordings using a Drummond microdispenser (Broomall, PA), respectively. Injected oocytes were incubated at 18°C for 3–5 days in sterile ND-96 medium. Immediately before patch-clamp experiments, the vitelline membrane was removed with fine forceps.

Electrophysiological recordings and data analysis
All single-channel and macroscopic current recordings were performed using the giga-ohm seal patch-clamp method in either excised inside-out or outside-out configuration. Patch pipettes were fabricated from borosilicate glass (WPI, Sarasota, FL) and fire polished to the resistance of 2–5 M for macroscopic patches and 5–8 M for single-channel recording. For single-channel recordings, patch pipettes were coated with beeswax. The channel currents were amplified using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA), low-pass filtered at 1 or 2 kHz using a four-pole Bessel filter, and digitized at a rate of 10 or 20 points/ms using Digidata 1200A (Axon Instruments). No series resistance compensation was used and linear leak currents were subtracted from macroscopic currents.

For single-channel recordings, membrane patches of inside-out configuration were held at various membrane potentials. Single rSlo or rSlo/rß4 channels were readily activated at high levels of intracellular Ca²⁺. For single-channel analysis, transitions between closed and open states were determined by setting the threshold at half of the unitary current amplitude. To determine the single-channel conductance of rSlo and rSlo/rß4 channels, mean amplitudes of channel currents were obtained from histograms fit with Gaussian distributions and the mean current levels were plotted against transmembrane voltages. Slope conductance value was obtained by linear regression. Dwell times of open and closed events recorded for single rSlo and rSlo/rß4 channels were analyzed using the logarithmic binning method. The dwell-time distributions were fitted with two exponentials using simplex-least-squares fitting methods (Fechan and pSTAT, Axon Instruments). The peaks in the dwell-time distributions fall at the time constants of each exponential component.

Macroscopic currents of the BK_Ca channel were activated by voltage pulses delivered from a holding potential of –100 mV to membrane potentials ranging from –140 to 140 mV in 10-mV or 20-mV increments. Recording solutions for both single and macroscopic channels expressed in oocytes contained gluconates as a nonpermeant anion to prevent the activation of endogenous calcium-activated chloride channels. The intracellular and extracellular solutions contained the following components unless specified otherwise (in mM): 120 potassium gluconate, 10 HEPES, 4 KCl, and 5 EGTA (pH 7.2). CaCl₂ was added into intracellular solution to provide the required free-Ca²⁺ concentration (0.1–20 µM) calculated using a stability constant for Ca-EGTA of 10.86, and the calculation included a pH adjustment (Martell and Smith, 1974). To compare the characteristics of the channels accurately, an identical set of intracellular solutions was used throughout the entire experiments: single and macroscopic current recordings, and the recordings of the channels composed of both rSlo alone and rSlo with rß4. Commercial software packages such as Clampex 8.0 or 8.1 (Axon Instruments), and Origin 6.1 (OriginLab, Northampton, MA) were used for the acquisition and the analysis of single-channel and macroscopic recording data.

Statistical analysis
All data were presented as means ± SE and n indicates the number of independent experiments. For each data set, the statistical significance of the difference was tested using ANOVA for independent observations. In all cases P < 0.01 was considered significant. Each macroscopic current trace represents an average of three records in succession.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Isolation of cDNA for ß4-subunit of rat BK_Ca channel and its molecular biological characterization
To obtain the cDNA of the BK_Ca channel ß4-subunit expressed in rat brain, we screened 2.7 x 10⁶ plaque-forming unit of rat total brain cDNA library and isolated a single clone covering the full length of the ß4 open reading frame. The full-length cDNA, named as rß4, was 1,028 bp with a 633-bp-long ORF and encodes a protein product of 210 amino acids (GenBank accession number AY028605). When compared with other family members of human BK_Ca channel ß-subunits, the deduced amino acid sequences of rß4 showed 18%, 25%, 22%, and 94% to hß1, hß2, hß3, and hß4, respectively. The open reading frame and deduced amino acid sequence of rß4 were compared to the previous reported orthologs of human ß4 (hß4, GenBank accession number AF207992) and mouse ß4 (mß4, GenBank accession number AF215892). The rß4 were 94% and 99% identical to hß4 and mß4, respectively. The amino acid sequence of rß4 and mß4 was identical except only a single residue at position 169 (valine for rß4 and alanine for mß4). The hydrophobicity analysis of the rß4-subunit using Kyte-Dolittle methods predicted two putative transmembrane regions and a large extracellular loop amino acid sequence (data not shown) as adopted in the previous study with mß4.

To check whether any alternative splicing variants of rß4 occur in different regions of rat brain, we prepared total RNAs from five distinct regions of rat brain (whole brain, cerebral cortex, mid brain, cerebellum, and brain stem) and performed reverse transcriptase-polymerase chain reaction (RT-PCR). We initially noticed that the open reading frame of the human ß4 gene is encoded by three different exons separated by 27- and 33-k basepairs in human chromosome 12q14.1–15. A pair of DNA primers was designed to recognize the entire open reading frame of rß4 coding regions. The RT-PCR resulted in single amplified DNA bands with an identical size in all five different subregions of the brain. The sequencing analysis of RT-PCR products also confirmed that only a single type of rß4 message identical to the clone we obtained was expressed in different regions of rat brain (data not shown).

Effects of rß4 on conductance-voltage relationship of rSlo channel
To investigate the detailed functional effects of rß4 on rSlo channels, cRNAs of rSlo and rß4 were synthesized in vitro and injected into Xenopus oocytes. We usually used 12-fold molar excess of the rß4 transcripts to ensure the sufficient coassembly of the rß4-subunit with the rSlo subunit. From 3 to 5 days after injection, macroscopic channel activities of rSlo and rSlo coexpressed with rß4 were measurable. Representative current traces from inside-out patch recordings are shown for rSlo (Fig. 1 A) and rSlo/rß4 channels (Fig. 1 B). Recording in low and high free calcium required different voltage steps for rSlo and rSlo/rß4 channels to reach the levels of steady-state activation. To obtain accurate maximum conductance and to compare distinct current characteristics between rSlo and rSlo/rß4, macroscopic rSlo and rSlo/rß4 channel currents were elicited with 50-ms, 100-ms, or 200-ms-long voltage steps in different intracellular calcium concentrations. The representative traces were obtained using voltage steps of 100 ms and 200 ms for rSlo and rSlo/rß4 channels, respectively. The activation of channel currents at various test potentials was measured using tail currents at 0.8 or 1 ms after repolarization to –100 mV. The activation of macroscopic currents of both rSlo and rSlo/rß4 channels was highly dependent on intracellular Ca²⁺ concentrations and membrane potentials.

View larger version (40K): [in this window] [in a new window]   FIGURE 1  Representative current traces from excised patches expressing rSlo and rSlo/rß4 channels and effects of rß4-subunit on the conductance-voltage relationship of rSlo channels. Macroscopic currents of rSlo (A) and rSlo/rß4 (B) were recorded in symmetrical 124 mM K+-gluconate. The concentrations of intracellular free Ca2+, [Ca2+]i, were indicated. Ionic currents were elicited with voltage steps of 100 ms for rSlo and 200 ms for rSlo/rß4 to test potentials ranging from –140 mV to 140 mV in 10-mV increments. The holding voltage was –100 mV. Each current trace represents an average of three records in succession. Tail currents of each test potential were determined at 0.8 or 1 ms after repolarization to –100 mV. The conductance-voltage (G-V) relationship for rSlo (C) and rSlo/rß4 (D) were shown. The concentrations of intracellular free calcium were shown as follows: Ca2+-free (squares), 0.5 µM (circles), 1 µM (diamonds), 1.4 µM (up triangles), 1.7 µM (down triangles), 2 µM (hexagons), 5 µM (asterisks), and 20 µM (pentagons). The solid lines represent the best fit of the VD-MWC model, Popen = 1/[1 + {(1 + [Ca2+]/KC)/(1 + [Ca2+]/KO)}4L(0)e–QFV/RT], proposed previously for mSlo channel (Cox and Aldrich, 2000). L(0) represents the open-to-closed equilibrium constant in the absence of an applied voltage, Q the equivalent gating charge associated with this equilibrium, KC the closed-conformation Ca2+ dissociation constant, and KO the open-conformation Ca2+ dissociation constant. Parameters of the fits are as follows: for rSlo, L(0) = 942.86 ± 36.89, Q = 1.04 ± 0.04, KO = 0.62 ± 0.06 µM, KC = 6.57 ± 0.48 µM; for rSlo/rß4, L(0) = 980, Q = 1.35, KO = 1.2 µM, KC = 0.6 µM at 0.5 µM [Ca2+]; L(0) = 980, Q = 1.35, KO = 0.66 µM, KC = 6.2 µM at 1 µM, 1.4 µM, and 1.7 µM [Ca2+]; L(0) = 600, Q = 1.37, KO = 0.42 µM, KC = 8.43 µM at 2 µM, 5 µM, and 20 µM [Ca2+]. (E) Relationship between intracellular Ca2+ concentration and the half-activation voltages. Each data point was obtained from the best fits of Boltzmann function, G/Gmax = 1/[1 + exp{zF(V1/2 – V)/RT}], from independent data sets. Data points for rSlo and rSlo/rß4 are denoted as open and solid symbols, respectively. Each data point represents the mean ± SE from at least 10 recordings of six different oocytes. Asterisks represent the significant differences tested using ANOVA test for independent observations (P < 0.01).

In a series of previous studies, a modified version of the Monod-Wyman-Changeux (MWC) model for allosteric proteins was applied successfully to explain gating properties of BK_Ca channels (Cui et al., 1997; Cox et al., 1997, Cox and Aldrich, 2000). Based on the "two-tiered gating scheme," Cox and Aldrich applied a voltage-dependent version of the MWC (VD-MWC) model to interpret the activation of mouse Slo (mSlo) (Cox and Aldrich, 2000). Although the model was simple and includes several assumptions, many aspects of the mSlo channel especially the effects of Ca²⁺ and voltage could be interpreted successfully. We applied the VD-MWC model to our experimental results and simulated the effects of rß4. The solid lines in Fig. 1, C and D, represent the best fit of a VD-MWC model coplotted with experimental data. The rß4-subunit increased the apparent affinity of the rSlo channel for Ca²⁺ >2 µM (the dissociation constant for Ca²⁺ in closed conformation, K_C: 6.57 ± 0.48 for rSlo, 8.4 ± 0.97 for rSlo/rß4; the dissociation constant for Ca²⁺ in open conformation, K_O: 0.62 ± 0.06 for rSlo, 0.42 ± 0.03 for rSlo/rß4). However, the apparent Ca²⁺ affinity of rSlo/rß4 was decreased dramatically at lower Ca²⁺ concentrations (K_C: 6.57 for rSlo, 0.6 for rSlo/rß4; K_O: 0.62 for rSlo, 1.2 for rSlo/rß4). At intermediate Ca²⁺ concentrations (e.g., 1.4 µM and 1.7 µM Ca²⁺), the rß4-subunit did not affect the Ca²⁺ affinity significantly (K_C: 6.57 for rSlo, 6.2 for rSlo/rß4; K_C: 0.62 for rSlo, 0.66 for rSlo/rß4).

In summary, the ß4-subunit increased the voltage range of the BK_Ca channel activation in a Ca²⁺-dependent manner. This effect of the ß4-subunit should be more prominent when the intracellular Ca²⁺ concentrations are relatively high. Although the rSlo channels evoke only 12% of maximal currents at –50 mV in the presence of 5 µM, the rSlo/rß4 channel can be activated to 45% under the identical conditions.

Effects of ß4-subunit on activation and deactivation kinetics of macroscopic rSlo channel currents
As shown in the raw traces of Fig. 1, A and B, the gating kinetics of macroscopic rSlo channel currents was significantly affected by the coexpression of the rß4-subunit. The ionic currents of the initial 100 ms and 200 ms activated by a step pulse from –140 mV to 140 mV were measured for the rSlo and rSlo/rß4 channels, respectively, at different [Ca²⁺]_i, and normalized to the maximum current levels for direct comparison (Fig. 2 A). The coexpression of rß4 decreased the activation rate of the rSlo channels at low intracellular Ca²⁺ such as at 0.5–1 µM (Fig. 2 A, left and middle panels). However, this effect of rß4 disappeared gradually as the concentration of intracellular Ca²⁺ was increased and the activation of rSlo became faster (Fig. 2 A, right panel). The activation time constants of the rSlo and rSlo/rß4 channel currents were estimated by fitting the current traces to single exponentials and the activation rates (1/) were plotted as a function of membrane potentials in Fig. 2 B. Not only the activation rates but also the voltage dependence of the rSlo channel activation were significantly altered. The activation rates of the rSlo/rß4 channel were less sensitive to the transmembrane voltages compared with those of rSlo at identical [Ca²⁺]_i and the trend was more dramatic as the concentration of Ca²⁺ was decreased. In fact, the activation of rSlo/rß4 was almost insensitive to transmembrane voltage at 0.5 µM and 1 µM of [Ca²⁺]_i ( Fig. 2 B, solid circles and triangles in right panel).

View larger version (30K): [in this window] [in a new window]   FIGURE 2  Activation and deactivation kinetics of rSlo and rSlo/rß4 channels. (A) Activation characteristics of rSlo and rSlo/rß4 channels. Channel currents of rSlo (black) and rSlo/rß4 (gray) activated by a step pulse from –100 mV to 100 mV were normalized with their maximum currents. (C) Deactivation characteristics of rSlo and rSlo/ß4 channels. Tail currents were elicited with 100-ms voltage steps to test potentials ranging from –100 mV to 20 mV in 20-mV increments from a prepulse at 100 mV. Tail currents were normalized with their maximum currents. Activation and deactivation rate (1/) of macroscopic rSlo (B) and rSlo/rß4 (D) were plotted as a function of membrane voltage. Activation and deactivation time constant () of macroscopic rSlo and rSlo/rß4 channel currents were obtained by fitting the individual current traces to simplex single exponential function. The concentrations of intracellular free calcium were as follows: 0.5 µM (circles), 1 µM (up triangles), 2 µM (down triangles), 5 µM (diamonds), and 20 µM (hexagons). Data points for rSlo and rSlo/rß4 are denoted as open and solid symbols, respectively. Each data point represents the mean ± SE from at least eight independent recordings.

Effects of ß4-subunit on Ca²⁺-dependent activation of single rSlo channels
To reveal the detailed functional effects of the rß4-subunit, rSlo and rSlo/rß4 channels were investigated at the single-channel level. To confirm the excised inside-out patches containing a single channel, we recorded the channel currents in extended periods at high levels of intracellular Ca²⁺ or depolarized potentials expected to activate readily the expressed channels. Single-channel recordings were performed in various durations for investigation of steady-state kinetics. We recorded for 3–5 min (up to 2000 of the channel transitions) at low Ca²⁺ concentrations and for 0.5–2 min (up to 10,000 of the channel transitions) at high levels of Ca²⁺. Representative traces of single rSlo (Fig. 3 A) and rSlo/rß4 channels (Fig. 3 B) were shown. The currents were recorded at 40 mV under various [Ca²⁺]_i. The openings of single rSlo and rSlo/rß4 channels were highly dependent on the intracellular Ca²⁺ concentrations as expected. Although the single-channel conductance of rSlo was not altered by the coexpression of rß4 (data not shown), the gating behavior of single rSlo/rß4 channels was significantly different from those of rSlo channels. The individual open events of the rSlo/rß4 channel recorded at low [Ca²⁺]_i was much longer compared with those of rSlo channels (see 1-µM traces in Fig. 3 shown in two different timescales). Especially, single-channel transitions of rSlo/rß4 channels were remarkably slower than rSlo channels at low Ca²⁺ such as 0.5 µM and 1 µM Ca²⁺.

View larger version (34K): [in this window] [in a new window]   FIGURE 3  Representative single-channel currents of rSlo and rSlo/rß4 expressed in Xenopus oocytes. Typical single-channel current recordings of rSlo (A) and rSlo/rß4 (B) channels were shown at different intracellular Ca2+ concentrations. Ionic currents of single channels in a patch of oocytes membrane were continuously recorded at 40 mV in different intracellular Ca2+ concentrations. Activity of the single channel was highly dependent on the concentration of intracellular Ca2+. The data were sampled at 10 kHz or 20 kHz and filtered at 2 kHz. Single-channel currents of rSlo and rSlo/rß4 were measured under identical recording solutions for macroscopic channel currents recording as previously shown in Fig. 1.

View larger version (27K): [in this window] [in a new window]   FIGURE 4  Comparison between open probability for single channels and activation of macroscopic currents for rSlo and rSlo/rß4 channels by Ca2+. Single-channel open probability of rSlo (A, ) and rSlo/rß4 (B, ) recorded with various intracellular Ca2+ concentrations were coplotted with the activation of macroscopic channel currents (rSlo, ; rSlo/rß4, ). Recording of both single and macroscopic currents were obtained at 40 mV. (C) Relationship of open probability against intracellular Ca2+ concentration of rSlo () and rSlo/rß4 () was fitted to the Hill equation. (D) Open probability single rSlo (open bars) and rSlo/rß4 (striped bars) determined at 0.5, 1, and 1.4 µM of intracellular Ca2+. The mean values of the open probability for each channel was given in the text. Identical recording solutions were used for both macroscopic and single-channel recordings. Each data point represents the mean ± SE from eight (for rSlo) and nine (for rSlo/rß4) independent recordings. Asterisks represent the significant differences tested using ANOVA test for independent observations#, (#, P < 0.05; *, P < 0.01).

View larger version (32K): [in this window] [in a new window]   FIGURE 5  Representative dwell-time histograms of single rSlo and rSlo/rß4 channels for open and closed events. Excised membranes were held at 40 mV in the presence of 0.5 µM (A), 1 µM (B), and 2 µM (C) intracellular Ca2+ concentrations. The histograms of both open and closed dwell times obtained from 2,000 (at low Ca2+ concentrations, continuously recorded for 3–5 min) to 10,000 (at high Ca2+ concentrations, continuously recorded for 0.5–2 min) transitions were plotted in log-bin timescales and fitted with a combination of two exponential functions. Solid lines on the bar graphs represent the sum of two exponential functions of time constants, 1 and 2.

View larger version (19K): [in this window] [in a new window]   FIGURE 6  Effects of rß4 on the steady-state gating kinetics of single rSlo channels. Time constants of open (A) and closed events (B) for rSlo () and rSlo/rß4 (•) were plotted as a function of intracellular Ca2+ concentrations. The time constants, 1 and 2, represent the faster and slower components of open and closed dwell times. Each data point represents the mean ± SE from at least six independent single-channel recordings. Asterisks represent the significant differences tested using ANOVA for independent observations (P < 0.01).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

Compared with the rSlo channel (or BK_Ca channels of homotetrameric -subunits) currents, the macroscopic currents of the rSlo/rß4 channel showed the marked differences in the conductance-voltage relationship, the gating kinetics, and the cooperativity for intracellular Ca²⁺. The activation of rSlo/rß4 channels was affected differently depending on the concentration of intracellular calcium. Although the conductance-voltage curves were shifted only slightly to more depolarizing voltages at low Ca²⁺ concentration, the G-V relationship shifted dramatically more negative potentials at high Ca²⁺ concentration. As the results, the BK_Ca channels composed of rSlo and rß4 can open at more hyperpolarized membrane voltages when the concentration of intracellular Ca²⁺ is progressively increased. Because the local concentration of intracellular Ca²⁺ is known to increase up to tens of micromolars during the action potential (Regehr and Tank, 1992), the half-activation voltages (V_1/2) are expected to shift >30 mV toward hyperpolarization direction. This result is also consistent with a previous report in which the effects of four different human ß-subunits on G-V curves were compared (Brenner et al., 2000).

It was shown that the ß1-subunit increased the apparent Ca²⁺ sensitivity by increasing open probability and shifted half-activation voltages to significantly more negative potentials (McManus et al., 1995; Knaus et al., 1994; Dworetzky et al., 1996; Cox and Aldrich, 2000; Nimigean and Magleby, 1999). Although the effect of rß4 on conductance-voltage curves is similar to that of ß1 at high Ca²⁺ concentrations, rß4 slightly but significantly changes the voltage dependence of rSlo channel at low Ca²⁺concentrations. The simulation of the VD-MWC model based on our experimental data provided valuable information on the effects of ß4 on the gating of the rSlo channel. Although the rß4-subunit increased the apparent affinity of the rSlo channel for Ca²⁺ >2 µM similar to the ß1-subunit (Cox and Aldrich, 2000), the apparent affinity of rSlo/rß4 for Ca²⁺ was decreased dramatically at lower Ca²⁺ concentrations. Therefore, the effects of the rß4-subunit are quite different from those of the ß1-subunit, because the ß1-subunit increases the Ca²⁺ affinity regardless of its concentration. The ß4-subunit increases the affinity for Ca²⁺ to the closed state of the Slo channel and thus makes the channel close at low Ca²⁺. In high Ca²⁺, however, the same subunit increases the affinity for Ca²⁺ to the open state and tends to open the channel. Thus, the ß4-subunit makes the correlation between the changes in V_1/2 and [Ca²⁺]_i and broadens the effective ranges of voltage-dependent activation under various intracellular Ca²⁺ concentrations (Fig. 1 E). In general, the neuronal BK_Ca channels containing the ß4-subunit would require significantly lower voltages to be activated at Ca²⁺ concentration >1 µM. Because the BK_Ca channels coassembled with ß4 exhibits higher cooperativity for Ca²⁺ (Fig. 4), the channels should be able to respond more sensitively to the increase in intracellular Ca²⁺. It is also intriguing that the same channels may not be activated efficiently under submicromolar concentrations of local Ca²⁺ under physiological transmembrane voltages (Fig. 4, C and D).

Functional rSlo channels are activated and deactivated rapidly in response to depolarizing and hyperpolarizing step pulses of membrane voltages. The kinetics of macroscopic currents becomes much slower when rSlo assembles with rß4. The kinetics of rSlo/rß4 channels was less dependent upon the membrane potential and significantly slower compared with those of rSlo. Thus, the slowing of macroscopic gating events may render significant physiological consequences. For an example, whereas the effects of the ß4-subunit on the activation of BK_Ca channels is minimal at 10 µM, the deactivation of channel current is more than twofold slower throughout the transmembrane voltage and thus the hyperpolarizing effects of the BK_Ca channel would be significantly longer. It remains to be determined how the gating effects of the ß4-subunit are represented in the firing pattern of neuronal action potentials and the modulation of neuroexcitability. The ß4-subunit also affects the steady-state gating behavior of BK_Ca channels. Although the rSlo channels showed rapid transitions between open and close states, much longer opening and closing events were observed for rSlo/rß4. The most dramatic effect of the ß4-subunit on the single BK_Ca channel was the decrease in closing and opening rates for the slow component at marginal concentration of Ca²⁺.

One of the most noticeable effects of the ß4-subunit revealed in this study was dramatic increase in the Ca²⁺ cooperativity for the BK_Ca channel. Although the Ca²⁺-dependnent activation of the rSlo channel can be fit with the Hill coefficient of 3.2, that of rSlo/rß4 was best fit with 6.5. The highly cooperative activation in the presence of the ß4-subunit makes the BK_Ca channel more sensitive to the concentration changes in intracellular Ca²⁺. About twofold increase in intracellular Ca²⁺ from 1 to 2 µM can result in almost full activation of BK_Ca channels composed of rSlo/rß4 and the effects can be solely explained with the increase in the open probability of single BK_Ca channels. Knowing the fact that the BK_Ca channel is likely composed of four -subunits, it is intriguing to find that the Hill coefficient for Ca²⁺ can be as high as about seven. It will be important to determine in the subsequent studies what is the molecular mechanism of this high Ca²⁺ cooperativity rendered by the ß4-subunit especially in the context of protein structure. In conclusion, the neuronal-specific ß-subunit of ß4 modulates the functional characteristics of the BK_Ca channel -subunits by altering not only the gating kinetics but also the sensitivity and the cooperativity for intracellular Ca²⁺. It would be required in future works to confirm the effects of the ß4-subunit on neuronal BK_Ca channels in vivo and to elucidate the functional roles of this auxiliary subunit on the excitability of neuronal cells in the brain in situ.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The authors thank Dr. Richard W. Aldrich at Stanford University for the cDNA of the human BK_Ca channel ß4-subunit. We also thank the other members of the Laboratory of Molecular Neurobiology at K-JIST for their valuable comments and timely help throughout the work. The nucleotide sequence of rat BK_Ca channel ß4-subunit reported in this article was deposited in the GenBank with accession number AY028605.

This research was supported by research grants from the Ministry of Science and Technology of Korea (Brain Neurobiology Research, M1-0108-00-005), the Korea Science and Engineering Foundation (R02-2000-00192), and the Korea Research Foundation (BK21) to C.-S. Park.

Submitted on December 3, 2003; accepted for publication January 12, 2004.

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