|
|
|
|
|
|
|
||
ARTICLE |
Subunits in the Ca2+-activated K+ (BK) Channel
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and ß1 or ß2 are dissimilar, the most remarkable difference being a reduction of the voltage sensitivity in the presence of ß1 but not ß2. Here we reveal the molecular regions in these ß subunits that determine their differential functional coupling with the pore-forming -subunit. We made chimeric constructs between ß1 and ß2 subunits, and BK channels formed by and chimeric ß subunits were expressed in Xenopus laevis oocytes. The electrophysiological characteristics of the resulting channels were determined using the patch clamp technique. Chimeric exchange of the different regions of the ß1 and ß2 subunits demonstrates that the NH3 and COOH termini are the most relevant regions in defining the behavior of either subunit. This strongly suggests that the intracellular domains are crucial for the fine tuning of the effects of these ß subunits. Moreover, the intracellular domains of ß1 are responsible for the reduction of the BK channel voltage dependence. This agrees with previous studies that suggested the intracellular regions of the -subunit to be the target of the modulation by the ß1-subunit.
P. Orio's present address is the Instituto de Neurociencias de Alicante, Universidad Miguel Hernandez-CSIC, 03550 Sant Joan d'Alacant, Spain.
P. Rojas's present address is the Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110
 |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; McManus and Magleby, 1991; Toro et al., 1998; Vergara et al., 1998). The BK channel is a homotetramer of its pore-forming -subunit, which is coded by the gene Slo1 (KNCMA1). This protein contains seven putative transmembrane segments (S0–S6) where the primary sequence of S1–S6 is homologous to the corresponding domains in KV channels (Shen et al., 1994; Meera et al., 1997). The channel is gated both by membrane depolarization and by increased intracellular Ca2+ concentration. Thorough studies of BK channel gating suggest that calcium and voltage act as allosteric modulators (Cox et al., 1997; Cox and Aldrich, 2000; Horrigan and Aldrich, 2002).
In some mammal tissues, BK channel subunits are coexpressed with modulatory ß subunits, four of which have been cloned (Knaus et al., 1994b; Jiang et al., 1999; Wallner et al., 1999; Xia et al., 1999; Behrens et al., 2000; Brenner et al., 2000; Meera et al., 2000; Uebele et al., 2000; for review see Orio et al., 2002). Purification and cloning of the ß1-subunit unveiled the presence of a protein containing two putative transmembrane regions joined together by a large extracellular loop (Knaus et al., 1994a,b). ß1 increases the apparent Ca2+ sensitivity of the channel, slows down the macroscopic activation and deactivation kinetics, and decreases the voltage sensitivity (McManus et al., 1995; Wallner et al., 1995; Dworetzky et al., 1996; Meera et al., 1996; Cox and Aldrich, 2000; Nimigean and Magleby, 2000; Bao and Cox, 2005; Orio and Latorre, 2005). The presence of the ß1-subunit is also a requirement for the binding of channel activators (McManus et al., 1995; Valverde et al., 1999; Dick et al., 2001). The S0 domain and the extracellular NH2 terminus of the -subunit are crucial for the adequate functional coupling between - and ß1-subunit (Wallner et al., 1996). The ß2-subunit has an N-type or fast inactivation motif in its NH2 terminus, causing BK currents to inactivate when this subunit is present (Wallner et al., 1999; Xia et al., 1999). This motif can be removed (ß2IR-subunit) and the activation properties of the channel can be studied in the presence of ß2IR without the contamination of the inactivation process. Like the ß1-subunit, ß2IR also increases the apparent Ca2+ sensitivity of the BK channels and slows down its macroscopic kinetics (Wallner et al., 1999; Xia et al., 1999). But unlike the ß1-subunit, ß2IR does not modify the voltage dependence and confers low affinity to CTX compared with that exhibited by the -subunit (Xia et al., 1999; Orio and Latorre, 2005).
Although the region in the -subunit responsible for ß1-subunit regulation has been mapped to the S0 membrane-spanning and NH2-terminal regions, very little is known about the structural determinants contained in the ß subunits that determine the functional coupling between and ß subunits. We showed in detail that ß1 and ß2IR subunits conferred different gating properties to the BK channel (Orio and Latorre, 2005). In particular, they impart to BK channels different calcium sensitivities, voltage dependences, and macroscopic gating kinetics. Here we use these differences to map the regions in the ß subunits responsible for ß-subunit regulation by constructing chimeras between ß1 and ß2IR subunits. Our results show that intracellular NH2 and COOH termini are responsible for most of the modulatory effects on channel activity, with some participation of the transmembrane regions. The extracellular regions have no participation in the differences between ß1 and ß2IR subunits and may play a structural function related to other effects of the ß subunits on the channel.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-subunit (KCNMA1) from myometrium (GenBank/EMBL/DDBJ accession no. U11058), human ß1 and ß2 subunits (KCNMB1 and KCNMB2, GenBank/EMBL/DDBJ accession no. U25138 and AF099137), and ß2-subunit without inactivation domain (ß2IR) (Wallner et al., 1999) were used. The boundaries of the transmembrane domains of ß1 and ß2IR subunits were defined according to Wallner et al. (1999). Chimeric ß subunits were made with the overlapping extension method (Horton et al., 1990) and confirmed by DNA sequencing.
Channel Expression
mMESSAGE mMACHINE (Ambion) in vitro transcription kit was used to obtain mRNAs. Channels were expressed in Xenopus oocytes using standard techniques (Stühmer and Parekh, 1995). -Subunit cRNA (0.75 to 2 ng) or a mixture of -subunit (0.5–1.5 ng) and ß-subunit (2–3 ng) cRNAs were injected per oocyte. The approximate molar ratio was at least 6:1 (ß:), ensuring saturation of ß-subunit.
Electrophysiological Recordings and Solutions
Currents were recorded 1–5 d after cRNA injection using the inside-out configuration of the patch-clamp technique. All recordings were performed at room temperature (20–22°C). Intracellular (bath) and extracellular (pipette) solutions contained (in mM) 110 KOH, 10 HEPES, and 2 KCl and were adjusted to pH 7.4 with methanosulfonic acid. Depending on the desired free calcium concentration, 1.8–3.5 mM CaCl2 and 5 mM EGTA (5–200 nM), HEDTA (0.6–12 µM), or NTA (20–150 µM) was added. Free calcium concentrations were calculated using the WinMaxChelator Software (http://www.stanford.edu/
cpatton/maxc.html) and checked with a calcium electrode (World Precision Instruments). The solution designated as "5 nM" is an upper estimation based on contaminating calcium. Depending on the magnitude of currents, and in order to minimize voltage drops due to series resistance, some experiments were done with solutions containing 36 mM KOH and 74 mM NMDG. The pipette solution contained 100 nM or lower free calcium.
Pipettes were pulled in a horizontal pipette puller (Sutter Instruments) from Corning 7740 (Pyrex) or Custom 8250 (Warner Instruments) borosilicate capillary glass. Pipette resistance was 0.8–2 M
in 110 mM K+, and series resistance error was always <10 mV.
Data were acquired with an Axopatch 200B (Axon Instruments) or an EPC-7 (List Medical) amplifier, filtered with an 8-pole Bessel filter (Frequency Devices) at 1/5 of the acquisition rate, and sampled with a 16-bit A/D converter (NI-6036, National Instruments). Typical acquisition rates ranged from 66.6 to 100 kHz (-subunit alone) or from 16.6 to 40 kHz ( + ß subunits). Homemade acquisition software was developed in the LabView programming environment (National Instruments). Primary data analysis was performed with Analysis (provided by F. Bezanilla, University of California Los Angeles, CA) and Clampfit 9 (Axon Instruments) software.
Steady-state Activation Analysis
The Solver function of Microsoft Excel was used to fit a Boltzmann function of the form
 | (1) |
Statistical Analysis
The 
act/V curves were compared by a two-way ANOVA test, thus assessing the statistical significance of the datasets as a whole. The V0.5/[Ca2+] curves could not be compared by a two way ANOVA test because not all the chimeras were studied at the same calcium concentrations. Instead, the statistical difference between two datasets was performed by testing whether they can be fit by two (independent fit) or a single (global fit) dose–response curve. Comparison between independent and global fits was done by an extra sum-of-squares F test, using the formula
 |
deact values in the –50 to 60 mV range. The z values showed the greatest variability of all the parameters studied, and the statistical comparisons were not reliable. All the analyses were done with the GraphPad Prism software (GraphPad Software Inc.). |
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) we showed clear differences in the way that the ß1 and ß2 subunits modulate BK channel's properties, suggesting a different mechanism of action. The differences in the modulation of steady-state activation of the channel are summarized in Fig. 1. As the ß2-subunit induces a fast or N-type inactivation of the current (Fig. 1 A, ß2wt), in the previous as well as in this work we studied a truncated ß2-subunit (
2-19ß2, ß2IR) that lacks the inactivation peptide. In the presence of the ß2IR-subunit, BK currents are sustained (Fig. 1 A, +ß2IR) and therefore the effects of this subunit in the activation parameters of the BK channel are best studied.
|
+ß1 and +ß2IR are lower than for at [Ca2+] 
1 µM. However, the half potential shift to more hyperpolarized voltages is more pronounced in + ß2IR channels. At [Ca2+] 
100 nM, +ß1 channels have higher values than either or +ß2IR channels while +ß2IR is not different from . Finally, the ß1-subunit produces a reduction in the BK voltage dependence, not observed when channels are formed by + ß2IR subunits (Fig. 1 D).
In the case of the kinetic parameters, both ß subunits slow down the activation kinetics. However, + ß2IR channels show a slower activation and the voltage dependence of the deactivation kinetics is modified by the presence of the ß1- but not the ß2-subunit (Orio and Latorre, 2005; see also Fig. 4, C and D).
|
+ ß1 and + ß2IR subunits. We, therefore, predicted that regions essential for ß-subunit modulation of the BK channel may be identified by exchanging regions between ß1 and ß2 subunits. The first set of chimeras tested was one in which the external loops were exchanged: ß2 containing the loop of ß1 (ß2Lß1) and ß1 containing the loop of ß2 (ß1Lß2) (Fig. 2 A). Fig. 2 B shows that coexpression of these chimeras with the -subunit produces robust currents in excised patches of oocyte membrane. In particular, it is apparent from the current records that both chimeras are able to slow down the activation and deactivation kinetics with respect to currents generated by -subunit alone (see also Fig. 4). Fig. 2 C shows that the conductance is voltage and Ca2+ dependent. A more detailed inspection indicates that the G/V curves for the ß2Lß1 chimera are steeper and that at 3 µM Ca2+ it is shifted 40 mV to the left compared with the ß1Lß2 chimera. To quantify the effects of each chimera and to have an adequate comparison of their characteristics with those of the parent ß subunits we performed experiments over a wide range of Ca2+ concentrations. Fig. 3 (A and B) summarizes the results obtained from the fitting of the G/V curves to Eq. 1. Fig. 3 A shows a plot of V0.5 against [Ca2+] for ß1Lß2 (black triangles) and ß2Lß1 (open triangles). Fig. 3 B shows the z values against [Ca2+] for ß1Lß2 (black triangles) and ß2Lß1 (open triangles). For visual comparison purposes, the data for , + ß1, and + ß2IR channels are shown only as the best fit curves (see figure legends for details). The results demonstrate that the behavior of channels formed by + ß1Lß2 resembles that of channels produced by the coexpression of + ß1 (P = 0.6 and 0.002 compared with + ß1 and + ß2IR, respectively, in an extra sum-of-squares F test; see MATERIALS AND METHODS) and that the activity of channels formed by + ß2Lß1 is similar to channels formed by + ß2IR (P = 0.002 and 0.72 compared with + ß1 and + ß2IR, respectively). This result suggests that the loop region is not responsible for the functional differences between ß1 and ß2 subunits.
|
|
+ ß2IR channels is slower than that promoted by + ß1 channels (Orio and Latorre, 2005). Fig. 4 A shows representative current traces for channel activation at 200 mV after a –80 hyperpolarizing pulse in the virtual absence of calcium (5 nM). The time course followed by the currents induced by + ß2Lß1 channels is slower than that of + ß1Lß2 channels. Fig. 4 C plots the time constant of an exponential fit of the current time course as a function of the activation voltage. The tendency ( + ß2Lß1 slower than + ß1Lß2 slower than ) is maintained at all the activation voltages explored, resembling the behavior of the ß1 and ß2IR subunits. As in the V0.5/[Ca2+] and z/[Ca2+] plots, the data obtained for + ß1Lß2 channels superimposes to that obtained for + ß1 channels (P = 0.13 and P < 0.0001 when compared with + ß1 and + ß2IR, respectively, in a two-way ANOVA test), and the data obtained for + ß2Lß1 channels is not different from that obtained for + ß2 channels (P < 0.0001 and P = 0.94 when compared with + ß1 and + ß2IR, respectively).
The deactivation process at –60 mV after a 200-mV activation pulse is also slower when the channel is coexpressed with the chimeric subunits ß1Lß2 and ß2Lß1 (Fig. 4 B). Moreover + ß1Lß2 is slower than + ß2Lß1, confirming that ß1Lß2 behaves like ß1 while ß2Lß1 is like ß2IR. However, the deactivation kinetics must be studied in a wide voltage range because the ß1-subunit also affects the voltage dependence of the deactivation process. Fig. 4 D plots the deactivation time constant (deact) in the –220 to +100 mV voltage range. The semilogarithmic deact/V plot shows two apparent slopes for all , + ß1, and + ß2IR channels. While the first slope (limiting slope for the voltage dependence of channel deactivation) is not affected by the type of ß-subunit, the second slope (z = 0.44 electronic equivalents for the -subunit alone) is severely affected by the ß1 (z = 0.25) but not the ß2IR (z = 0.44) subunit. This is due to the reduction of the voltage sensor–associated voltage dependence of the BK channel that occurs in the presence of the ß1-subunit (Orio and Latorre, 2005; but see Bao and Cox, 2005). The chimeric ß1Lß2-subunit modifies the voltage dependence of the deactivation process of the channel in the same way as the ß1-subunit, with a z value of 0.28 (P = 0.8 and P < 0.0001 compared with + ß1 and + ß2IR, respectively, in an extra sum-of- squares F test). The ß2Lß1-subunit shows a voltage sensor–related slope a little higher than that observed for + ß2IR channels (z = 0.48, P < 0.0001 and P = 0.003 compared with + ß1 and + ß2IR, respectively).
These results strongly suggest that the differential effects of the ß subunits on the BK channel properties are related to the transmembrane and/or the intracellular domains of these regulatory subunits but not to the extracellular domain.
Functional Coupling Is Mainly Determined by Intracellular Domains
To circumscribe the structure(s) in the ß subunits determining the differential coupling with the -subunit, we exchanged the NH2 and COOH terminus between ß1 and ß2IR. The two chimeras generated in this way were denominated ß1NCß2 (ß1 with NH2 and COOH termini of ß2) and ß2NCß1 (ß2 with NH2 and COOH termini of ß1) (Fig. 5 A). Fig. 5 B shows that the coexpression of both chimeras with the BK channel produces robust, calcium- and voltage-dependent currents. From the G/V plots (Fig. 5 C), it is evident that in the presence of the ß1NCß2 chimera, the channel has higher calcium sensitivity than in the presence of ß2NCß1 (the curve for 3 µM is more displaced to the left). Also, + ß1NCß2 channels have a higher voltage dependency. The analysis of the calcium and voltage dependence (Fig. 6, A and B) in the presence of these chimeras shows that the ß2NCß1 chimera (filled triangles) imparts to the BK channel similar properties than those obtained when the -subunit is coexpressed with the ß1-subunit (black solid line), both in terms of the V0.5/[Ca2+] (P = 0.03 and P < 0.0001 compared with + ß1 and + ß2IR, respectively) as well as the z/[Ca2+] relationships. The ß1NCß2 chimera (empty triangles) on its hand resembles the behavior of the ß2IR (gray solid lines) subunit, though the V0.5 values are displaced 40–50 mV toward more negative voltages (P < 0.0001 and P = 0.003 compared with + ß1 and + ß2IR, respectively).
|
|
act) at several voltages and in the absence of calcium. ß1NCß2 resembles the behavior of the ß2IR-subunit (P < 0.0001 and P = 0.08, respectively) and though the ß2NCß1 is statistically different from both (P = 0.0002 and P < 0.0001, respectively), it is closer to the ß1-subunit. This suggests that the intracellular portions of the ß subunits are responsible for the modulation of the activation kinetics.
In the case of the macroscopic deactivation kinetics (Fig. 6 D), none of the chimeras behaved exactly as either the ß1- or the ß2IR-subunit. The voltage dependence of the deactivation kinetics turned out to be different to both parent ß subunits (z = 0.35 ± 0.02 for + ß2NCß1 and 0.36 ± 0.02 for + ß1NCß2 channels; P 0.01 for all comparisons with + ß1 and + ß2IR), suggesting a partial effect on the voltage sensor–associated voltage dependence. + ß1NCß2 channels have deact limiting values (at the most negative potentials) close to + ß1 channels (P = 0.03), indicating that the kinetic rate for channel deactivation when all voltage sensors are in the resting position is affected by this chimera in the same way as in the presence of the ß1-subunit.
In summary, the study of the "NC" chimeras suggests that the intracellular regions of the ß1 and ß2IR subunits determine the modulation of a great part of the properties studied here, with the exception of the changes induced in the deactivation kinetics and its voltage dependence. Essentially the same conclusion can be obtained from the analysis of the ß1TMß2 and ß2TMß1 chimeras, in which the transmembrane domains were exchanged between ß1 and ß2IR (Figs. 7 and 8). ß2TMß1 chimera, which contains the same intracellular domains as ß2 and ß1NCß2 subunits, behaves very much like the ß1NCß2 subunits. On the other hand, the ß2TMß1 chimera, which contains the same intracellular domains as the ß1 and ß2NCß1 subunits, behaves as the ß2NCß1 chimera. This can be seen from another point of view: the ß1NCß2 and ß2TMß1 chimeras modulate BK channel properties in a very similar way and they share the transmembrane and intracellular domains with a completely different extracellular loop. The same is true for the ß2NCß1 and ß1TMß2 chimeras. This is in complete agreement with the results of the ß1Lß2 and ß2Lß1 chimeras, as the extracellular loop does not determine the differential behavior of the ß subunits.
|
|
|
|
+ ß1Nß2 channels are very similar to + ß2IR in terms of the V0.5 (Fig. 9 B, P = 0.02 and P = 0.1 compared with + ß1 and + ß2IR, respectively), z (Fig. 9 C), and act (Fig. 9 D, P < 0.0001 and P = 0.6, respectively) values. The only reminiscences of the ß1-subunit are found in the deact/V plot, where the voltage sensor–associated voltage dependence (z = 0.28, P = 0.02 and P < 0.0001, respectively) and the limiting deact value are similar to + ß1. Something similar occurs with the opposite chimera, ß2Nß1, with some minor differences. + ß2Nß1 channels behave like + ß1 in terms of the z (Fig. 9 C) and act (Fig. 9 D, P = 0.5 and P < 0.0001, respectively) values; however, the V0.5 values fall between the curves for alone and + ß1 channels (Fig. 9 B; P = 0.001 when compared with + ß1 and 0.07 when compared with ). The latter may be an indication of a disruption of the effect of the ß subunits on the apparent calcium sensitivity. Regarding the deactivation kinetics, the behavior is intermediate between and + ß2IR channels, with a lower limiting deact than + ß2IR and a slope for the –50 to +50 voltage range of 0.34 (P < 0.01 when compared with both + ß1 and + ß2IR).
When the COOH termini of the ß1 and ß2IR subunits are transplanted between each other, the resulting chimeras have almost all the properties of the parent ß-subunit. The + ß1Cß2 channels have V0.5 values (P = 0.05 and P < 0.0001), z values, act values (P = 0.3 and P < 0.0001), and deact voltage dependence of + ß1 (Fig. 10, B–E); only the limiting deact resembles that of + ß2IR (Fig. 10 E; P = 0.2). + ß2Cß1 channels, on the other hand, behave like + ß2IR in terms of the z (Fig. 10 C), act (Fig. 10 D; P < 0.0001 and P = 0.07, respectively), and deact values (Fig. 10 E; P < 0.0001 and P = 0.3, respectively, for the –50 to 50 mV slope). Interestingly, the V0.5 values are like + ß1 (P = 0.7 and P = 0.0001, respectively), suggesting again a partial disruption of the normal effect of the ß2IR-subunit on the apparent calcium sensitivity.
In summary, a gross analysis of the results with the "N" and "C" chimeras indicates that most of the differences between ß1 and ß2IR subunits reside in the corresponding NH2 terminus, and that for some of the effects (changes in deact, for example) both intracellular domains are required.
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
–ß Subunit Coupling-subunit together with the first transmembrane domain S0 have been proposed to contribute to the functional interaction between ß and subunits (Wallner et al., 1996). This conclusion was put forward based on the results obtained using different chimeras between hSlo and the Drosophila homologue of hSlo, dSlo, that does not functionally couple to the ß1-subunit. A chimera containing only the extracellular NH2 terminus together with the first transmembrane domain S0 of hSlo is able to successfully couple with the ß1-subunit. Here we attempt to draw similar conclusions from the side of the ß1 and ß2 subunits, characterizing a set of chimerical ß subunits. A general view of our results shows that the intracellular regions of ß1 and ß2 are responsible for most of the differences between the modulatory effects that each of them confer to the activity of BK channel.
The ß1 and ß2 subunits are the most closely related within the BK channel's ß-subunit family, with a 43% overall sequence identity and a 39% identity in the loop region. Leaving aside the inactivation promoted by the ß2-subunit, they have qualitatively similar effects on BK channel properties. Our conclusions, therefore, should be drawn strictly on the differences between these subunits. It is possible that most of the effects of both subunits reside in a common structural feature of the extracellular loop, while the intracellular and transmembrane domains would be responsible for the fine tuning of these effects giving to each subunit its own identity. Though this is an issue that will need experiments with the other ß subunits to be categorically addressed, several facts make us think that these domains do much more than subtle changes to the activation properties of the BK channel. First, one of the differences between ß1 and ß2 subunits is not quantitative but qualitative: the voltage dependence of the channel is greatly impaired in the presence of the ß1-subunit while it remains unaffected by the ß2-subunit. At least, our present results map regions of the ß1-subunit that must interact with either the voltage sensors of the channel or the domains that couple the voltage sensor activation with channel opening. Second, a thorough analysis of the effects of these subunits suggested the possibility that both ß subunits enhance the apparent calcium sensitivity by different mechanisms (Orio and Latorre, 2005). In this case, the regions that we have mapped would be responsible for a different regulatory mechanism in each ß-subunit. Finally, all the voltage and calcium sensitivity in the BK channel has been so far mapped to transmembrane and intracellular domains: the voltage sensor is in the S4 membrane–spanning helix (Diaz et al., 1998), all the postulated calcium binding sites are proposed to be intracellular (Schreiber and Salkoff, 1997; Schreiber et al., 1999; Bian et al., 2001; Bao et al., 2002, 2004; Xia et al., 2002, 2004), and the coupling mechanisms for both voltage and calcium activation have been mapped to intracellular regions as well (see below, Functional Coupling). Therefore we think unlikely that an important contribution to the modulation of calcium- and voltage-dependent activation mechanisms of the BK channel may arise from an interaction of extracellular domains of ß and subunits.
Function of the Extracellular Region
Several roles have been attributed to the interaction between extracellular regions of the and ß subunits of the BK channel, all of them unrelated to changes in the voltage or calcium sensitivity of the channel. The external loop of ß subunits plays an important role in determining ion permeation and the characteristics of toxin binding. The ß4-subunit, for instance, induces a resistance of BK channels to CTX and IbTX block that is reproduced only by a chimera containing the loop of ß4 (Meera et al., 2000). The ß3-subunit produces an open channel rectification (Xia et al., 2000; Lingle et al., 2001; Zeng et al., 2001) that is dependent on the disulfide bridges in its extracellular region. Again, this effect is only reproduced by a chimera containing the extracellular loop of ß3 (Zeng et al., 2003). In the case of the bovine ß1-subunit, the adequate loop conformation appears to be a requisite for BK channel high affinity binding of CTX, as any of the four cysteines contained in the external ß1 linker are critical in defining the ability of ß1 in enhancing CTX binding (Hanner et al., 1998). Regarding the functional coupling between and ß1 subunits, the results that propose the extracellular domains as part of the interaction domain (Wallner et al., 1996) may be only of structural meaning, these domains being needed for the assembly of the heteromultimer rather than for functional coupling.
The present results add to this list of findings that the ß-subunit external region may play only a minor role in the modulation of the calcium- and voltage-dependent activation mechanisms of the channel. This minor contribution cannot be excluded, though. We have recently shown that a point mutation in the extracellular loop of the human ß1-subunit modifies its effect on BK calcium sensitivity (Fernandez-Fernandez et al., 2004). Despite the physiological relevance of this effect, it is less than the differences between ß1 and ß2 subunits (e.g., at 500 nM calcium the E65K mutation induces a shift of 10 mV in V0.5 while the difference between ß1 and ß2 subunits at 680 nM calcium is 20 mV), and this mutant still changes the channel kinetics the way the wild-type ß1-subunit does. Thus, the extracellular loop may play a partial role in the modulation of apparent calcium sensitivity, possibly via some interaction with the external side of the voltage sensor (see Bao and Cox, 2005; Orio and Latorre, 2005).
Role of the Intracellular Termini
When only the intracellular and transmembrane regions are dissociated (NC and TMs chimeras) the resulting chimeras induce changes in channel properties that resemble the donor of the intracellular regions, suggesting that the intracellular ends are responsible for most of the functional coupling between and ß subunits. However in this case the results were not as clean as with the L chimeras, suggesting a shared role with the transmembrane regions in the modulation of channel properties. Interestingly, the ß1NCß2 and ß2TMsß1 chimeras (both having the NH2 and COOH termini of ß2) showed an enhanced effect on BK channel's calcium sensitivity, promoting a V0.5 shift higher than that promoted by the ß2IR-subunit.
Chimeras with only the NH2 terminus exchanged (ß1Nß2 and ß2Nß1) showed a mixed behavior; however, there were important characteristics similar to the ß-subunit contributing to the NH2 terminus. In particular, the NH2 terminus suffices to determine the BK channel activation kinetics and the behavior of z as a function of the Ca2+ concentration. A different result was obtained with the chimeras with only the COOH terminus exchanged (ß1Cß2 and ß2Cß1); in which there were minor differences in behavior compared with the parental ß subunits. Thus, the NH2 terminus of the ß subunits is the most important structural determinant in the differences of modulation of the -subunit by its auxiliary ß1 and ß2 subunits.
This finding is not surprising when other evidences are considered. In the case of ß4-subunit, phosphorylation of its NH2 and COOH termini is crucial for the functional coupling with the -subunit (Jin et al., 2002). The addition of the phosphatase inhibitor okadaic acid (OA) eliminates the effect of coexpression of ß4 subunits, indicating that phosphorylation of ß4 eliminates its modulatory effects. This shows that the relevance of the intracellular ends of the ß subunits in the –ß subunit coupling may be a characteristic of all ß subunits. The NH2 terminus of ß2-subunit has a conserved cAMP, cGMP-dependent phosphorylation site (sequence KRKT), and its relevance for modulation of channel properties remains unknown.
Functional Coupling
It has been proposed that the intracellular domains of each BK -subunit contributes two RCK (regulatory of K+ conductance) domains and that these eight RCK domains form a gating ring similar to the gating ring of MthK channels (Jiang et al., 2001, 2002). The free energy provided by Ca2+ binding to the gating ring (chemical energy) is transformed in mechanical energy used to open the pore. The linker between the sixth (S6) transmembrane domain and the first RCK domain of the BK channel has also been shown to play a role in this molecular coupling, acting as a passive spring (Niu et al., 2004). In the case of the molecular coupling between voltage sensor activation and channel opening, the S4–S5 linker as well as the COOH- terminal half of the S6 segment have been postulated to play this role in other voltage-gated channels (Chen et al., 2001; Lu et al., 2002; Tristani-Firouzi et al., 2002).
It appears, therefore, inescapable that the intracellular domains of the BK channel are the most probable site of interaction for the modulation of the calcium and voltage sensitivity. Indeed, Qian et al. (2002) showed that the ß1-subunit cannot enhance the calcium sensitivity of BK channels when the intracellular "tail" domain is replaced with the tail from the highly related, pH-dependent, Slo3 channel. The only effect of the ß1-subunit that can be detected on this channel is a reduction of the voltage dependence as it does for the wt channel in the absence of calcium. This and other observations were interpreted as the ß1-subunit interacting with the intracellular calcium-sensing machinery of the BK channel. We show here that the intracellular domains of the ß1 and ß2 subunits are indeed responsible for most of the channel activity modulation. An attractive hypothesis would be that the cytoplasmic regions of the ß subunits interacts directly with the linker-gating ring complex, modifying the allosteric coupling factors involved in channel opening.
|
ACKNOWLEDGMENTS |
|---|
This work was supported by grants from the Fondo Nacional de Investigación Científica y Tecnológica (FONDECYT 103-0830 to R. Latorre, 200-0061 to P. Orio, and 201-0006 to P. Rojas), the Human Frontiers in Science Program (R. Latorre, M.A. Valverde, and L. Toro), National Institutes of Health (HL54970 to L. Toro), and Red HERACLES (FIS, Spain, to M.A. Valverde). During part of this work, P. Orio was recipient of a Ph.D. fellowship from Fundación Andes. The Centro de Estudios Cientificos is a Millenium Institute and is funded in part by a grant from Fundacion Andes.
Olaf S. Andersen served as editor.
Submitted: 26 July 2005
Accepted: 13 January 2006
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  T.-B. Sweet and D. H. Cox Measuring the Influence of the BKCa {beta}1 Subunit on Ca2+ Binding to the BKCa Channel J. Gen. Physiol., February 1, 2009; 133(2): 139 - 150. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Liu, S. I. Zakharov, L. Yang, R. S. Wu, S.-X. Deng, D. W. Landry, A. Karlin, and S. O. Marx Locations of the {beta}1 transmembrane helices in the BK potassium channel PNAS, August 5, 2008; 105(31): 10727 - 10732. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Lingle Mg2+-dependent Regulation of BK Channels: Importance of Electrostatics J. Gen. Physiol., December 31, 2007; 131(1): 5 - 11. [Full Text] [PDF]
|
|
|
|
 |
|
 Y. P. Torres, F. J. Morera, I. Carvacho, and R. Latorre A Marriage of Convenience: beta-Subunits and Voltage-dependent K+ Channels J. Biol. Chem., August 24, 2007; 282(34): 24485 - 24489. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Savalli, A. Kondratiev, S. B. de Quintana, L. Toro, and R. Olcese Modes of Operation of the BKCa Channel {beta}2 Subunit J. Gen. Physiol., July 1, 2007; 130(1): 117 - 131. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Y. Kim, S. Zou, L. D. Ridgway, and S. E. Dryer beta1-Subunits Increase Surface Expression of a Large-Conductance Ca2+-Activated K+ Channel Isoform J Neurophysiol, May 1, 2007; 97(5): 3508 - 3516. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
