Mutation of slo Thr107 modifies channel ethanol responses
Primary sequence alignment of bslo and mslo showed that nonconserved regions occurred both in the S0–S1 loop and the S8–S9 linker. Considering that the vast majority of voltage-gated K+
channels are refractory to modulation by clinically relevant concentrations of ethanol29,30
and that these channels lack the S0 segment, we mutated the threonine present in the S0–S1 loop that is not conserved between bslo and mslo.
Oocyte expression of bslo, mslo, bslo T107V () or mslo V86T led to single-channel events that showed all the major characteristics of BK currents when recorded in inside-out (I/O) membrane patches using symmetric 130 mM K+
solutions (Methods). First, at fixed free internal calcium concentrations ([Ca2+
; 300 nM), the voltage–steady-state activity (that is, NPo
; see Methods) relationship of both mutant channels was described by a Boltzmann function in which the plot of ln(NPo
) versus voltage was linear at low NPo
. Thus, the inverse of the slope (k
) was the potential needed to produce an e
-fold change in NPo
): 13 ± 3 mV (n
= 6) and 16 ± 3 mV (n
= 6) for bslo T107Vand mslo V86T, respectively. These values are similar (P
> 0.05) and fall within the range reported for mslo, bslo and other slo channels11,15,17
. Second, the NPo
of both mutant channels obtained at any given voltage increased similarly with increases in [Ca2+
(data not shown). Finally, both mutant channels showed similar high unitary conductance: 225 ± 23 pS and 208 ± 18 pS (P
> 0.05; n
= 6) in symmetric 130 mM K+
for bslo T107V and mslo V86T, respectively. These values are similar to those reported for mslo and bslo channels under equivalent conditions11,15
. These similarities in k
activation and unitary conductance reflect the high identity that exists in the amino acid sequences of the voltage-sensing Ca2+
-bowl and pore regions across slo subunits2
; further, they indicate that the mutations did not modify critical aspects of BK channel function.
Figure 2 Ethanol at clinically relevant concentrations increases bslo T107V channel activity (NPo) after expression of these slo subunits in Xenopus oocytes. (a–c) Traces showing single channel recordings from the same I/O membrane patch obtained before (more ...)
Acute ethanol administration (100 mM) reversibly increased mslo channel NPo
in ten of ten I/O patches. In contrast, ethanol evoked varied responses in bslo channels: decreased activity in eight, increased activity in four and no change in four. After data from all samples passed a Kolmogorov-Smirnov normality test (P
> 0.1), parametric analysis determined that the averaged ethanol responses of mslo and bslo (196 ± 16.67% and 106 ± 11.3% of pre-ethanol controls) were different (P
< 0.05), even when studied in the same batches of oocytes (). These data confirm previous findings12
and support the idea that regions that are not conserved between these slo subunits are major determinants of the differential BK channel responses to ethanol.
Figure 3 The valine-to-threonine mutation in the slo S0–S1 loop significantly modifies BK channel responses to acute ethanol exposure. (a) Average changes in channel NPo in response to ethanol are shown as mean ± s.e.m. where n (number of patches (more ...)
Ethanol readily and reversibly increased bslo T107V channel activity without modifying current amplitude (). Thus, drug action seems to be limited to modulation of channel gating. Ethanol, however, increased channel NPo
without modifying k: k
was 13 ± 3 mV versus 12 ± 4 mV per e
-fold change in NPo
in the presence and absence of ethanol, respectively (n
= 6). Under steady-state conditions, an increase in NPo
may be determined by an increase in channel mean open time, a decrease in mean closed time or both. Ethanol did not change the mean open time, which was 2.6 ± 0.3 ms in the absence of ethanol versus 2.5 ± 0.2 ms in the presence of ethanol (P
> 0.05; n
= 5). Thus, ethanol potentiation of bslo T107V channels was caused by an increase in the frequency of channel openings (that is, decrease in channel mean closed time). These results are similar to those obtained with mslo15
and native BK channels in neurohypophysial nerve endings4
Ethanol-induced increases in bslo T107V NPo were observed in 12 of 12 patches (). Thus, for both bslo T107V and mslo, ethanol increased channel NPo in all patches tested (n = 22). Not only the frequency of bslo T107V channel activity, but also its average potentiation (247.8 ± 15.3% of controls), were similar to those found with mslo (P > 0.05) (). In contrast, bslo T107V NPo responses to ethanol were markedly different from bslo responses (P < 0.001). These data demonstrate that the single mutation of threonine to valine is sufficient to modify bslo channel responses to acute ethanol exposure.
In sharp contrast to its consistent activation of mslo and bslo T107V channels, ethanol had varied effects on mslo V86T channel activity: decreases (eight patches), increases (eight patches) and no change (three patches), a pattern similar to that of bslo channels (). Furthermore, the average NPo of mslo V86T in the presence of ethanol reached 123.63 ± 13.44% of that in controls, a value similar to that of bslo (P > 0.05) but different from that of mslo (P < 0.001) (). These data seem to indicate that the presence of a valine residue in the S0–S1 loop ‘locks’ slo channels into (or shifts the channel population to) a state that is readily activatable by ethanol.
Protein phosphatase switches channel responses to ethanol
We used a sequence- and structure-based program for predicting eukaryotic protein phosphorylation sites (NetPhos 2.0 server) and found that slo Thr107, in contrast to valine, has a rather high probability (≥0.4) of being phosphorylated. This raised the possibility that different degrees of phosphorylation of Thr107 determine, or at least contribute to, the heterogeneity of bslo responses to alcohol.
Slo subunits contain several sites that may be phosphorylated by various cross-talking signaling molecules22
; this makes it difficult to determine, a priori
, which phosphorylating signal should be probed so as to test the role of slo phosphorylation in modulating channel responses to ethanol. Thus, we first investigated whether nonselective dephosphorylation could lock slo channels containing Thr107 into an ethanol-activatable state. We treated I/O patches with alkaline phosphatase to dephosphorylate the bslo subunit (and nearby associated proteins; see Methods).
Alkaline phosphatase treatment increased channel activity to 192.6% of the values before alkaline phosphatase (n
= 10), as reported with native BK channels in bovine tracheal myocytes31
. These channels contain slo subunits identical in sequence to those used in our study. Thus, accessory β1
-subunits, which are tightly associated with bslo in the native channel2
but absent in our expression system11
, seem to be unnecessary for alkaline phosphatase to modulate bovine tracheal BK channels.
Before alkaline phosphatase treatment, the NPo responses of bslo channels to ethanol were typically heterogeneous and could be grouped into potentiation (that is, ‘activatable channel population’; n = 5) and refractoriness or inhibition (that is, ‘nonactivatable channel population’; n = 5) (). In this nonactivatable population, ethanol responses averaged 83.7 ± 9.2% of pre-ethanol controls. In sharp contrast, after alkaline phosphatase incubation of the same excised patches for 5 min, both activatable and nonactivatable bslo channels were activated by ethanol (), with overall NPo in the presence of ethanol averaging 242.3 ± 22.8% of that in the pre-ethanol controls (P < 0.01). This qualitative switch in the ethanol responses of bslo channels cannot be explained by ethanol modification of alkaline phosphatase activity, as ethanol was applied to the patch >5 min after phosphatase was removed from the bath. Rather, the switch in ethanol responses of bslo channels by alkaline phosphatase is probably a result of dephosphorylation of a target in the membrane patch, perhaps the slo subunit itself.
In vitro treatment with alkaline phosphatase shifts wild-type bslo channels into a state that is readily activatable by ethanol, but does not modify bslo T107V channel responses to alcohol
Notably, after alkaline phosphatase treatment, ethanol activation of the previously nonactivatable population of channels was similar to its effect on the activatable population before the application of alkaline phosphatase (P > 0.05) (). Furthermore, alkaline phosphatase did not modify ethanol responses in the activatable population of channels (). Thus, channels characterized by an ethanol response that was unmodified by alkaline phosphatase seem to remain in an ethanol-activatable state before being exposed to the phosphatase.
In contrast to the bslo results, alkaline phosphatase did not modify bslo T107V ethanol responses; NPo reached 223 ± 24.6% and 229.6 ± 18.9% of pre-ethanol controls (P > 0.05; n = 7) before and after dephosphorylating treatment. Therefore, alkaline phosphatase–mediated dephosphorylation probably modifies bslo channel responses to ethanol by targeting Thr107. The results from the alkaline phosphatase experiments on bslo and bslo T107V support the idea that the dephosphorylation of Thr107 locks the channel population into a state or states readily activatable by ethanol. Next, we explored which phosphorylation signaling molecule was involved in modulating ethanol action.
CaMKII modifies BK activity and its modulation by ethanol
The sequence KEETV in the bslo S0–S1 loop is similar to RQES/TV, a CaMKII conserved motif28
. Thus, we hypothesized that CaMKII phosphorylation before patch excision from the cell modifies slo Thr107 and, thus, channel responses to ethanol.
We first determined whether CaMKII could be detected in the intact cell under our recording conditions. Western analysis using polyclonal antibodies (Santa Cruz Biotechnology) identified an ~50-kDa band corresponding to CaMKII (J.L., A.M.D. and S. Tavalin, unpublished observations). This finding is consistent with a previous report identifying CaMKII in Xenopus
. Next, we used KN-93, a rather selective CaMKII inhibitor33
, to probe any involvement of the CaMKII phosphorylation of Thr107 in slo channel responses to ethanol.
After cells were incubated with KN-93 (20 μM for 15 min), all bslo channels were activated by ethanol, with average NPo increasing to 242.3 ± 12.9% of pre-ethanol controls (n = 9; ). This response was markedly different from the heterogeneous pattern and from the average response evoked by ethanol in the absence of KN-93 (P < 0.001; n = 16). Furthermore, ethanol responses of bslo channels in the presence of KN-93 were similar to the responses evoked by ethanol in mslo and bslo T107V (P > 0.05; ), both channels lacking the phosphorylatable Thr107 in the S0–S1 loop.
Figure 5 In vitro treatment with KN-93, a selective CaMKII inhibitor, shifted wild-type bslo channels into a state that is readily activatable by ethanol, but it does not modify bslo T107V channel responses to alcohol. Acute exposure of the cytosolic side of I/O (more ...)
In contrast, KN-92, an analog of KN-93 that does not inhibit CaMKII, failed to shift the bslo channel population to a state that was readily activatable by ethanol (n = 9). Indeed, ethanol responses of bslo channels in KN-92 were similar to those in the absence of CaMKII inhibitor (P > 0.05; ). Moreover, the ethanol responses of bslo channels in the presence of KN-92 were different from the ethanol responses of bslo channels in the presence of KN-93 (), and also from the responses of mslo and bslo T107V channels (P < 0.01).
In contrast to the results obtained with bslo, the ethanol responses of bslo T107V channels were similar before and after KN-93 treatment (P >0.05) ( and ). Together, these results strongly suggest that CaMKII modification of the ethanol responses of slo channels is a result of the phosphorylation of slo Thr107 by the kinase. Data obtained with selective inhibitors strongly suggest that endogenous basal CaMKII activity in the intact cell modulates ethanol responses of BK channels. We repeated these experiments with the different channel isoforms and mutants under a variety of treatments ().
Ethanol responses for different slo channel populations and treatments
To investigate, more directly, the role of CaMKII in modulating the ethanol responses of slo channels, we activated CaMKII in vitro with ATP + calmodulin and applied this phosphorylating complex (activated CaMKII + ATP + calmodulin) to I/O patches expressing bslo. After 5 min of incubation, we washed the patch in a complex-free bath solution for >5 min and then evaluated the action of ethanol on bslo channel currents. The application of CaMKII + ATP + calmodulin increased bslo channel currents to ~167% of those in the control (n = 5; , top panels), whereas the application of ATP + calmodulin repeatedly failed to increase current (n = 6). In contrast to bslo data, CaMKII + ATP + calmodulin did not modify bslo T107V currents (95.7 ± 16.4% of values before applying the phosphorylating complex; n = 6). Together these results indicate that CaMKII potentiates bslo channel currents by phosphorylating slo Thr107.
Figure 6 CaMKII increases wild-type bslo channel activity and switches channel responses to ethanol from activation to inhibition. (a,b) Current traces evoked at different potentials from excised I/O patches expressing wild-type bslo channels before (top), during (more ...)
To further evaluate a direct role of CaMKII-mediated phosphorylation in the alcohol responses of BK channels, we next focused on the ethanol-activatable bslo channel population. Currents from this population () were typically inhibited by ethanol after CaMKII phosphorylation treatment when evaluated in the same I/O patch (). This result () was replicated in four other patches, with average responses before () and after () CaMKII treatment shown as G
. The data demonstrate that neither ethanol nor CaMKII modified the voltage needed to produce an e
-fold change in G
(given by k
, the slope of the fit). CaMKII shifted the V0.5
of the G
relationship to the left: V0.5
= 122.8 ± 8.3 mVand 102.8 ± 6.8 mV in the absence and presence, respectively, of activated CaMKII (P
Ethanol substantially shifted the V0.5
to the left before CaMKII treatment () and to the right after CaMKII treatment () (all k
values are detailed in Supplementary Note
online). In brief, both CaMKII and ethanol modified bslo currents by producing a parallel shift in the G
relationship. These data suggest that both modulators modify channel steady-state activity without altering the number of charges mobilized across the electric field to gate the channel.
The results clearly demonstrate that the ethanol potentiation of bslo currents is switched to ethanol inhibition after CaMKII phosphorylating treatment (). This switch in ethanol responses is a mirror image of that caused by alkaline phosphatase () and KN-93 (). Furthermore, as previously found with alkaline phosphatase or KN-93 treatments ( and ), CaMKII+ATP+calmodulin failed to modify the ethanol responses of bslo T107V channels, which reached 104.8 ± 12.8% of controls (n = 6). Together these data suggest that it was the phosphorylation status of Thr107, secondary to CaMKII action, that determined the alcohol responses of slo channels.
CaMKII works as a molecular dimmer switch
The results seem to indicate that ethanol inhibits slo channels containing phosphorylated Thr107 while robustly activating channels containing dephosphorylated Thr107. Because BK channels are tetramers, the presence or absence of phosphate in different subunits could influence ethanol action, producing intermediate responses between robust potentiation and inhibition. Alternatively, the channel might respond to ethanol with a switch from robust activation to inhibition when a critical number of subunits is phosphorylated. To distinguish between these possibilities, we combined site-directed mutagenesis of Thr107 (to valine) with the mutation Y315V. The latter mutation in the extracellular vestibule of the channel () introduced resistance to external block by tetraethylammonium (TEA) in the assembled tetramer. Thus, by injecting oocytes with different molar ratios of bslo to bslo T107V Y315V (Methods), we determined the channel stoichiometry from measurements of unitary current amplitude in 2 mM external TEA, as previously described34
. After establishing a correspondence between channel stoichiometry and current amplitude (Supplementary Figure 1
online) and taking advantage of channel open probability (Po
) determinations from all-points amplitude histograms15
, we evaluated the action of ethanol on channel Po
from tetramers made of different subunit combinations.
Channels constructed with different combinations of bslo and bslo T107V Y315V were treated with CaMKII + ATP + calmodulin (see above), and unitary currents were evoked before (, left traces) and after (, right traces) ethanol exposure. As expected, these traces showed a progressive reduction in unitary current amplitude in the presence of TEA as the number of bslo T107V Y315V subunits was decreased from four (top row) to one (fourth row). In all patches, CaMKII failed to modify TEA action (data not shown).
Figure 7 CaMKII phosphorylation of Thr107 progressively switches channel responses to ethanol. (a) Current traces from tetramers made of wild-type bslo and bslo T107V Y315V combinations recorded before and after ethanol administration, after CaMKII treatment. (more ...)
From patches containing a single channel of a given subunit combination, we could determine Po
values, following CaMKII treatment, before and after ethanol application. This condition (that is, N
= 1) was very difficult to obtain owing to the well-known clustering of slo channels in the cell membrane11,15
. In four of four patches, the data obtained at constant transmembrane voltage (60 mV) and [Ca2+
(0.3 μM) indicated that the incremental phosphorylation, by CaMKII, of Thr107 in the bslo tetramer progressively increased basal Po
: the Po
values were 0.010 ± 0.007, 0.023 ± 0.004, 0.058 ± 0.006, 0.108 ± 0.005 and 0.218 ± 0.006 (mean ± s.e.m.; n
= 4) for tetramers having zero, one, two, three or four phosphorylated Thr107 subunits, respectively (, left traces). This increase in channel steady-state activity is likely a primary contributor to the increase in macroscopic current caused by CaMKII treatment ( versus 6b, top traces).
Unitary current records showed the characteristic ethanol inhibition of channel activity after CaMKII treatment (, bottom traces), with average values reaching 72.4 ± 8.4% of control (ethanol responses from bslo channels were evaluated in the absence of TEA because this agent totally blocked bslo currents, as has been reported with mslo channels34
). This ethanol decrease in Po
probably explains the ethanol inhibition of macroscopic current (). Alcohol inhibition of channel activity was lost when only one bslo T107V Y315V subunit was present in the tetramer; after CaMKII treatment, Po
in the presence of ethanol reached ~130% that of controls (, fourth traces from top, and , second column). Thus, ethanol inhibition of slo channels requires that all subunits in the tetramer be phosphorylated at Thr107. Our data indicate that CaMKII-mediated phosphorylation of this residue works as a molecular switch that shifts slo channel responses to ethanol from mild activation to inhibition ().
The robust potentiation by ethanol of homotetramers containing un-phosphorylatable residues (valine) in the S0–S1 loop gradually decreased as bslo T107V Y315V subunits were progressively replaced by slo subunits with phosphorylated Thr107 (). Average values for ethanol potentiation were 262.1 ± 16.1%, 200.1 ± 16.4%, 157.3 ± 10.8% and 120.1 ± 7.9% of pre-ethanol controls for tetramers having zero, one, two and three phosphorylated T107 subunits, respectively. Notably, the rate at which the ethanol potentiation of bslo channels changed with varying stoichiometry was constant (), which suggested that increasing the number of dephosphorylated subunits did not introduce cooperativity in ethanol-induced channel activation. In conclusion, CaMKII phosphorylation of Thr107 works as a molecular dimmer switch, progressively diminishing the activating effect of alcohol and finally switching it to inhibition.
Mutations to negatively charged residues, such as aspartate and glutamate, have been used to imitate a phosphorylated residue in proteins. Owing to steric and intrinsic activity differences, however, these mutations do not always produce a functional correlate in the protein that exactly mimics the effects of a phosphorylated residue35,36
We mutated Thr107 to a phosphomimetic glutamate and studied ethanol action on bslo T107E channels. Glutamate was chosen over aspartate because it is widely accepted that the extra methylene group of glutamate provides a chain length that more closely resembles the side chain of a phosphorylated threonine.
In response to ethanol, the NPo of bslo T107E channels reached 132.4 ± 6.1% of controls (n = 11). As expected for a phosphomimetic mutation, this response was different from that of bslo T107V in the same batch of oocytes (247.8 ± 17.7% of controls; P < 0.001; ). Although not identical to the ethanol responses of bslo in the presence of CaMKII, the ethanol responses of bslo T107E homotetramers (, middle column) were statistically indistinguishable (P > 0.05) from those evoked in heterotetramers containing two bslo T107V Y315V subunits (, third column). Thus, slo tetramers having a different subunit composition but the same number of negative charges in S0–S1 (that is, four) respond similarly to ethanol. In brief, the results obtained with bslo T107E partially mimicked the ethanol responses evoked in CaMKII-treated bslo channels, which is consistent with the idea that glutamate partially mimics a phosphorylated threonine. The results obtained with phosphomimetic bslo T107E support our conclusion that the phosphorylation of Thr107 progressively diminishes ethanol potentiation of BK channels.