Lysine-rich extracellular rings formed by hbeta2 subunits confer the outward rectification of BK channels

The auxiliary beta subunits of large-conductance Ca(2+)-activated K(+) (BK) channels greatly contribute to the diversity of BK (mSlo1 alpha) channels, which is fundamental to the adequate function in many tissues. Here we describe a functional element of the extracellular segment of hbeta2 auxiliary subunits that acts as the positively charged rings to modify the BK channel conductance. Four consecutive lysines of the hbeta2 extracellular loop, which reside sufficiently close to the extracellular entryway of the pore, constitute three positively charged rings. These rings can decrease the extracellular K(+) concentration and prevent the Charybdotoxin (ChTX) from approaching the extracellular entrance of channels through electrostatic mechanism, leading to the reduction of K(+) inflow or the outward rectification of BK channels. Our results demonstrate that the lysine rings formed by the hbeta2 auxiliary subunits influences the inward current of BK channels, providing a mechanism by which current can be rapidly diminished during cellular repolarization. Furthermore, this study will be helpful to understand the functional diversity of BK channels contributed by different auxiliary beta subunits.     

Species-specific Differences among KCNMB3 BK beta3 auxiliary subunits: some beta3 N-terminal variants may be primate-specific subunits

The KCNMB3 gene encodes one of a family of four auxiliary beta subunits found in the mammalian genome that associate with Slo1 alpha subunits and regulate BK channel function. In humans, the KCNMB3 gene contains four N-terminal alternative exons that produce four functionally distinct beta3 subunits, beta3a-d. Three variants, beta3a-c, exhibit kinetically distinct inactivation behaviors. Since investigation of the physiological roles of BK auxiliary subunits will depend on studies in rodents, here we have determined the identity and functional properties of mouse beta3 variants. Whereas beta1, beta2, and beta4 subunits exhibit 83.2%, 95.3%, and 93.8% identity between mouse and human, the mouse beta3 subunit, excluding N-terminal splice variants, shares only 62.8% amino acid identity with its human counterpart. Based on an examination of the mouse genome and screening of mouse cDNA libraries, here we have identified only two N-terminal candidates, beta3a and beta3b, of the four found in humans. Both human and mouse beta3a subunits produce a characteristic use-dependent inactivation. Surprisingly, whereas the hbeta3b exhibits rapid inactivation, the putative mbeta3b does not inactivate. Furthermore, unlike hbeta3, the mbeta3 subunit, irrespective of the N terminus, mediates a shift in gating to more negative potentials at a given Ca(2+) concentration. The shift in gating gradually is lost following patch excision, suggesting that the gating shift involves some regulatory process dependent on the cytosolic milieu. Examination of additional genomes to assess conservation among splice variants suggests that the putative mbeta3b N terminus may not be a true orthologue of the hbeta3b N terminus and that both beta3c and beta3d appear likely to be primate-specific N-terminal variants. These results have three key implications: first, functional properties of homologous beta3 subunits may differ among mammalian species; second, the specific physiological roles of homologous beta3 subunits may differ among mammalian species; and, third, some beta3 variants may be primate-specific ion channel subunits.     

Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels

An important step to understanding ion channels is identifying the structural components that act as the gates to ion movement. Here we describe a new channel gating mechanism, produced by the beta3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming alpha subunits. BK beta subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the beta3 subunits form gates to block ion permeation, providing a mechanism by which current can be rapidly diminished upon cellular repolarization. Furthermore, this gating mechanism is abolished by reduction of extracellular disulfide linkages, suggesting that endogenous mechanisms may regulate this gating behavior. The results indicate that auxiliary beta subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the alpha subunits to influence or block access of small molecules to the permeation pathway.     

The glycosylation of the extracellular loop of β2 subunits diversifies functional phenotypes of BK Channels

Large-conductance Ca²⁺- and voltage-activated potassium (MaxiK or BK) channels are composed of a pore-forming α subunit (Slo) and 4 types of auxiliary β subunits or just a pore-forming α subunit. Although multiple N-linked glycosylation sites in the extracellular loop of β subunits have been identified, very little is known about how glycosylation influences the structure and function of BK channels. Using a combination of site-directed mutagenesis, western blot and patch-clamp recordings, we demonstrated that 3 sites in the extracellular loop of β2 subunit are N-glycosylated (N-X-T/S at N88, N96 and N119). Glycosylation of these sites strongly and differentially regulate gating kinetics, outward rectification, toxin sensitivity and physical association between the α and β2 subunits. We constructed a model and used molecular dynamics (MD) to simulate how the glycosylation facilitates the association of α/β2 subunits and modulates the dimension of the extracellular cavum above the pore of the channel, ultimately to modify biophysical and pharmacological properties of BK channels. Our results suggest that N-glycosylation of β2 subunits plays crucial roles in imparting functional heterogeneity of BK channels, and is potentially involved in the pathological phenotypes of carbohydrate metabolic diseases.     

Functional roles of the extracellular segments of the sodium channel alpha subunit in voltage-dependent gating and modulation by beta1 subunits.

Voltage-gated sodium channels consist of a pore-forming alpha subunit associated with beta1 subunits and, for brain sodium channels, beta2 subunits. Although much is known about the structure and function of the alpha subunit, there is little information on the functional role of the 16 extracellular loops. To search for potential functional activities of these extracellular segments, chimeras were studied in which an individual extracellular loop of the rat heart (rH1) alpha subunit was substituted for the corresponding segment of the rat brain type IIA (rIIA) alpha subunit. In comparison with rH1, wild-type rIIA alpha subunits are characterized by more positive voltage-dependent activation and inactivation, a more prominent slow gating mode, and a more substantial shift to the fast gating mode upon coexpression of beta1 subunits in Xenopus oocytes. When alpha subunits were expressed alone, chimeras with substitutions from rH1 in five extracellular loops (IIS5-SS1, IISS2-S6, IIIS1-S2, IIISS2-S6, and IVS3-S4) had negatively shifted activation, and chimeras with substitutions in three of these (IISS2-S6, IIIS1-S2, and IVS3-S4) also had negatively shifted steady-state inactivation. rIIA alpha subunit chimeras with substitutions from rH1 in five extracellular loops (IS5-SS1, ISS2-S6, IISS2-S6, IIIS1-S2, and IVS3-S4) favored the fast gating mode. Like wild-type rIIA alpha subunits, all of the chimeric rIIA alpha subunits except chimera IVSS2-S6 were shifted almost entirely to the fast gating mode when coexpressed with beta1 subunits. In contrast, substitution of extracellular loop IVSS2-S6 substantially reduced the effectiveness of beta1 subunits in shifting rIIA alpha subunits to the fast gating mode. Our results show that multiple extracellular loops influence voltage-dependent activation and inactivation and gating mode of sodium channels, whereas segment IVSS2-S6 plays a dominant role in modulation of gating by beta1 subunits. Evidently, several extracellular loops are important determinants of sodium channel gating and modulation.     

Lysine-Rich Extracellular Rings Formed by hβ2 Subunits Confer the Outward Rectification of BK Channels

The auxiliary β subunits of large-conductance Ca²⁺-activated K⁺ (BK) channels greatly contribute to the diversity of BK (mSlo1 α) channels, which is fundamental to the adequate function in many tissues. Here we describe a functional element of the extracellular segment of hβ2 auxiliary subunits that acts as the positively charged rings to modify the BK channel conductance. Four consecutive lysines of the hβ2 extracellular loop, which reside sufficiently close to the extracellular entryway of the pore, constitute three positively charged rings. These rings can decrease the extracellular K⁺ concentration and prevent the Charybdotoxin (ChTX) from approaching the extracellular entrance of channels through electrostatic mechanism, leading to the reduction of K⁺ inflow or the outward rectification of BK channels. Our results demonstrate that the lysine rings formed by the hβ2 auxiliary subunits influences the inward current of BK channels, providing a mechanism by which current can be rapidly diminished during cellular repolarization. Furthermore, this study will be helpful to understand the functional diversity of BK channels contributed by different auxiliary β subunits.     

Interaction sites between the Slo1 pore and the NH2 terminus of the beta2 subunit, probed with a three-residue sensor

Calcium- and voltage-gated (BK) K(+) channels encoded by Slo1 play an essential role in nervous systems. Although it shares many common features with voltage-dependent K(V) channels, the BK channel exhibits differences in gating and inactivation. Using a mutant in which FWI replaces three residues (FIW) in the NH(2) terminus of wild-type beta2-subunits, in conjunction with alanine-scanning mutagenesis of the Slo1 S6 segment, we identify that the NH(2) terminus of beta2-subunits interacts with the residues near the cytosolic superficial mouth of BK channels during inactivation. The cytosolic blockers did not share the sites with NH(2) terminus of beta2-subunits. A novel blocking-inactivating scheme was proposed to account for the observed non-competition inactivation. Our results also suggest that the residue Ile-323 plays a dual role in interacting with the NH(2) terminus of beta2-subunits and modulating the gating of BK channels.     

Modes of operation of the BKCa channel beta2 subunit

The beta(2) subunit of the large conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca)) modulates a number of channel functions, such as the apparent Ca(2+)/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the beta(2) subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BK(Ca) channels (hSlo), we have investigated the mechanisms of operation of the beta(2) subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with beta(2) subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the beta(2) subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BK(Ca) channels coexpressed with the beta(2) subunit. The experimental observations are consistent with the view that the beta(2) subunit of BK(Ca) channels facilitates channel activation by changing the voltage sensor equilibrium and that the beta(2)-induced inactivation process does not follow a typical N-type mechanism.     

Structural basis for toxin resistance of beta4-associated calcium-activated potassium (BK) channels

The functional diversity of large conductance Ca(2+)- and voltage-dependent K(+) (BK) channels arises mainly from co-assembly of the pore-forming mSlo alpha subunits with four tissue-enriched auxiliary beta subunits. The structural basis of the interaction between alpha subunits with beta subunits is not well understood. Using computational and experimental methods, we demonstrated that four mSlo turrets decentralized distally from the channel pore to provide a wide open conformation and that the mSlo and hbeta4 subunits together formed a "helmet" containing three basic residues (Lys-120, Arg-121, and Lys-125), which impeded the entry of charybdotoxin (ChTX) by both the electrostatic interaction and limited space. In addition, the tyrosine insert mutant (in100Y) showed 56% inhibition, with a K(d) = 17 nm, suggesting that the hbeta4 lacks an external ChTX-binding site (Tyr-100). We also found that mSlo had an internal binding site (Tyr-294) in the alpha subunits that could "permanently" block 15% of mSlo+hbeta4 currents in the presence of 100 nm ChTX. These findings provide a better understanding of the diverse interactions between alpha and beta subunits and will improve the design of channel inhibitors.     

Gating properties conferred on BK channels by the beta3b auxiliary subunit in the absence of its NH(2)- and COOH termini

Both beta1 and beta2 auxiliary subunits of the BK-type K(+) channel family profoundly regulate the apparent Ca(2)+ sensitivity of BK-type Ca(2)+-activated K(+) channels. Each produces a pronounced leftward shift in the voltage of half-activation (V(0.5)) at a given Ca(2)+ concentration, particularly at Ca(2)+ above 1 microM. In contrast, the rapidly inactivating beta3b auxiliary produces a leftward shift in activation at Ca(2)+ below 1 microM. In the companion work (Lingle, C.J., X.-H. Zeng, J.-P. Ding, and X.-M. Xia. 2001. J. Gen. Physiol. 117:583-605, this issue), we have shown that some of the apparent beta3b-mediated shift in activation at low Ca(2)+ arises from rapid unblocking of inactivated channels, unlike the actions of the beta1 and beta2 subunits. Here, we compare effects of the beta3b subunit that arise from inactivation, per se, versus those that may arise from other functional effects of the subunit. In particular, we examine gating properties of the beta3b subunit and compare it to beta3b constructs lacking either the NH(2)- or COOH terminus or both. The results demonstrate that, although the NH(2) terminus appears to be the primary determinant of the beta3b-mediated shift in V(0.5) at low Ca(2)+, removal of the NH(2) terminus reveals two other interesting aspects of the action of the beta3b subunit. First, the conductance-voltage curves for activation of channels containing the beta3b subunit are best described by a double Boltzmann shape, which is proposed to arise from two independent voltage-dependent activation steps. Second, the presence of the beta3b subunit results in channels that exhibit an anomalous instantaneous outward current rectification that is correlated with a voltage dependence in the time-averaged single-channel current. The two effects appear to be unrelated, but indicative of the variety of ways that interactions between beta and alpha subunits can affect BK channel function. The COOH terminus of the beta3b subunit produces no discernible functional effects.     

Direct observation of a preinactivated, open state in BK channels with beta2 subunits

Proteins arising from the Slo family assemble into homotetramers to form functional large-conductance, Ca2+- and voltage-activated K+ channels, or BK channels. These channels are also found in association with accessory beta subunits, which modulate several aspects of channel gating and expression. Coexpression with either of two such subunits, beta2 or beta3b, confers time-dependent inactivation onto BK currents. mSlo1+beta3b channels display inactivation that is very rapid but incomplete. Previous studies involving macroscopic recordings from these channels have argued for the existence of a second, short-lived conducting state in rapid equilibrium with the nonconducting, inactivated conformation. This state has been termed "pre-inactivated," or O*. beta2-mediated inactivation, in contrast, occurs more slowly but is virtually complete at steady state. Here we demonstrate, using both macroscopic and single channel current recordings, that a preinactivated state is also a property of mSlo1+beta2 channels. Detection of this state is enhanced by a mutation (W4E) within the initial beta2 NH2-terminal segment critical for inactivation. This mutation increases the rate of recovery to the preinactivated open state, yielding macroscopic inactivation properties qualitatively more similar to those of beta3b. Furthermore, short-lived openings corresponding to entry into the preinactivated state can be observed directly with single-channel recording. By examining the initial openings after depolarization of a channel containing beta2-W4E, we show that channels can arrive directly at the preinactivated state without passing through the usual long-lived open conformation. This final result suggests that channel opening and inactivation are at least partly separable in this channel. Mechanistically, the preinactivated and inactivated conformations may correspond to binding of the beta subunit NH2 terminus in the vicinity of the cytoplasmic pore mouth, followed by definitive movement of the NH2 terminus into a position of occlusion within the ion-conducting pathway.     

Rectification ratio based determination of disulfide bonds of β2 extracellular loop of BK channel

Large-conductance Ca²⁺-activated K⁺ (BK) channels are composed of a pore-forming α and a variable number of auxiliary β subunits and play important roles in regulating excitability, action potential waveforms and firing patterns, particularly in neurons and endocrine and cardiovascular cells. The β2 subunits increase the diversity of gating and pharmacological properties. Its extracellular loop contains eight cysteine residues, which can pair to form a high-order structure, underlying the stability of the extracellular loop of β2 subunits and the functional effects on BK channels. However, how these cysteines form disulfide bonds still remains unclear. To address this, based on the fact that the rectification and association of BK α to β2 subunits are highly sensitive to disruption of the disulfide bonds in the extracellular loop of β2, we developed a rectification ratio based assay by combining the site-directed mutagenesis, electrophysiology and enzymatic cleavage. Three disulfide bonds: C1(C84)-C5(C113), C3(C101)-C7(C148) and C6(C142)-C8C(174) are successfully deduced in β2 subunit in complex with a BK α subunit, which are helpful to predict structural model of β2 subunits through computational simulation and to understand the interface between the extracellular domain of the β subunits and the pore-forming α subunit.     

Differential effects of beta 1 and beta 2 subunits on BK channel activity

High conductance, calcium- and voltage-activated potassium (BK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (beta) subunits. beta1 and beta2 subunits increase apparent channel calcium sensitivity. The beta1 subunit also decreases the voltage sensitivity of the channel and the beta2 subunit produces an N-type inactivation of BK currents. We further characterized the effects of the beta1 and beta2 subunits on the calcium and voltage sensitivity of the channel, analyzing the data in the context of an allosteric model for BK channel activation by calcium and voltage (Horrigan and Aldrich, 2002). In this study, we used a beta2 subunit without its N-type inactivation domain (beta2IR). The results indicate that the beta2IR subunit, like the beta1 subunit, has a small effect on the calcium binding affinity of the channel. Unlike the beta1 subunit, the beta2IR subunit also has no effect on the voltage sensitivity of the channel. The limiting voltage dependence for steady-state channel activation, unrelated to voltage sensor movements, is unaffected by any of the studied beta subunits. The same is observed for the limiting voltage dependence of the deactivation time constant. Thus, the beta1 subunit must affect the voltage sensitivity by altering the function of the voltage sensors of the channel. Both beta subunits reduce the intrinsic equilibrium constant for channel opening (L0). In the allosteric activation model, the reduction of the voltage dependence for the activation of the voltage sensors accounts for most of the macroscopic steady-state effects of the beta1 subunit, including the increase of the apparent calcium sensitivity of the BK channel. All allosteric coupling factors need to be increased in order to explain the observed effects when the alpha subunit is coexpressed with the beta2IR subunit.     

Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.     

Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel

We report here a characterization of two families of calcium-activated K(+) channel beta-subunits, beta2 and beta3, which are encoded by distinct genes that map to 3q26.2-27. A single beta2 family member and four alternatively spliced variants of beta3 were investigated. These subunits have predicted molecular masses of 27. 1-31.6 kDa, share approximately 30-44% amino acid identity with beta1, and exhibit distinct but overlapping expression patterns. Coexpression of the beta2 or beta3a-c subunits with a BK alpha-subunit altered the functional properties of the current expressed by the alpha-subunit alone. The beta2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the beta3a-c subunits resulted in only partial inactivation of the current, and the beta3b subunit conferred an apparent inward rectification. Furthermore, unlike the beta1 and beta2 subunits, none of the beta3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these beta-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.     

Steady-state and closed-state inactivation properties of inactivating BK channels

Calcium-dependent potassium (BK-type) Ca2+ and voltage-dependent K+ channels in chromaffin cells exhibit an inactivation that probably arises from coassembly of Slo1 alpha subunits with auxiliary beta subunits. One goal of this work was to determine whether the Ca2+ dependence of inactivation arises from any mechanism other than coupling of inactivation to the Ca2+ dependence of activation. Steady-state inactivation and the onset of inactivation were studied in inside-out patches and whole-cell recordings from rat adrenal chromaffin cells with parallel experiments on inactivating BK channels resulting from cloned alpha + beta2 subunits. In both cases, steady-state inactivation was shifted to more negative potentials by increases in submembrane [Ca2+] from 1 to 60 microM. At 10 and 60 microM Ca2+, the maximal channel availability at negative potentials was similar despite a shift in the voltage of half availability, suggesting there is no strictly Ca2+-dependent inactivation. In contrast, in the absence of Ca2+, depolarization to potentials positive to +20 mV induces channel inactivation. Thus, voltage-dependent, but not solely Ca2+-dependent, kinetic steps are required for inactivation to occur. Finally, under some conditions, BK channels are shown to inactivate as readily from closed states as from open states, indicative that a key conformational change required for inactivation precedes channel opening.     

Structural determinants for functional coupling between the beta and alpha subunits in the Ca2+-activated K+ (BK) channel

High conductance, calcium- and voltage-activated potassium (BK, MaxiK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (beta) subunits. The most remarkable effects of beta1 and beta2 subunits are an increase of the calcium sensitivity and the slow down of channel kinetics. However, the detailed characteristics of channels formed by alpha and beta1 or beta2 are dissimilar, the most remarkable difference being a reduction of the voltage sensitivity in the presence of beta1 but not beta2. Here we reveal the molecular regions in these beta subunits that determine their differential functional coupling with the pore-forming alpha-subunit. We made chimeric constructs between beta1 and beta2 subunits, and BK channels formed by alpha and chimeric beta 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 beta1 and beta2 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 beta subunits. Moreover, the intracellular domains of beta1 are responsible for the reduction of the BK channel voltage dependence. This agrees with previous studies that suggested the intracellular regions of the alpha-subunit to be the target of the modulation by the beta1-subunit.     

Endothelial K(+) channel lacks the Ca(2+) sensitivity-regulating beta subunit.

Hyperpolarizing large-conductance, Ca(2+)-activated K(+) channels (BK) are important modulators of vascular smooth muscle and endothelial cell function. In vascular smooth muscle cells, BK are composed of pore-forming alpha subunits and modulatory beta subunits. However, expression, composition, and function of BK subunits in endothelium have not been studied so far. In patch-clamp experiments we identified BK (283 pS) in intact endothelium of porcine aortic tissue slices. The BK opener DHS-I (0.05-0.3 micromol/l), stimulating BK activity only in the presence of beta subunits, had no effect on BK in endothelium whereas the alpha subunit selective BK opener NS1619 (20 micromol/l) markedly increased channel activity. Correspondingly, mRNA expression of the beta subunit was undetectable in endothelium, whereas alpha subunit expression was demonstrated. To investigate the functional role of beta subunits, we transfected the beta subunit into a human endothelial cell line (EA.hy 926). beta subunit expression resulted in an increased Ca(2+) sensitivity of BK activity: the potential of half-maximal activation (V(1/2)) shifted from 73.4 mV to 49.6 mV at 1 micromol/l [Ca(2+)](i) and an decrease of the EC(50) value for [Ca(2+)](i) by 1 microM at +60 mV was observed. This study demonstrates that BK channels in endothelium are composed of alpha subunits without association to beta subunits. The lack of the beta subunit indicates a substantially different channel regulation in endothelial cells compared to vascular smooth muscle cells.     

Interacting effects of N-terminal variation and strex exon splicing on slo potassium channel regulation by calcium,phosphorylation, and oxidation

We have investigated the structural basis for the phenotype of a native rat Slo (rSlo) potassium channel (BK(Ca); KCNMA1) in a rat pituitary cell line, GH(4)C(1). Opposing regulation of these calcium- and voltage-activated potassium channels by cAMP- and cGMP-dependent protein kinases requires an alternatively spliced exon (strex) of 59 amino acids in the cytoplasmic C terminus of the pore-forming alpha subunit encoded by rslo. However, inclusion of this cysteine-rich exon produces a 10-fold increase in the sensitivity of the channels to inhibition by oxidation. Inclusion of the strex exon also increases channel sensitivity to stimulation by calcium, but responses in the physiological ranges of calcium and voltage require coassembly with beta(1) subunits. With strex present, however, beta(1) subunits only stimulated channels assembled from rSlo alpha subunits with a truncated N terminus beginning MDALI-. Thus N-terminal variation and strex exon splicing in rSlo interact to produce BK(Ca) channels with a physiologically relevant phenotype.     

Inactivation of BK channels by the NH2 terminus of the beta2 auxiliary subunit: an essential role of a terminal peptide segment of three hydrophobic residues

An auxiliary beta2 subunit, when coexpressed with Slo alpha subunits, produces inactivation of the resulting large-conductance, Ca(2+) and voltage-dependent K(+) (BK-type) channels. Inactivation is mediated by the cytosolic NH(2) terminus of the beta2 subunit. To understand the structural requirements for inactivation, we have done a mutational analysis of the role of the NH(2) terminus in the inactivation process. The beta2 NH(2) terminus contains 46 residues thought to be cytosolic to the first transmembrane segment (TM1). Here, we address two issues. First, we define the key segment of residues that mediates inactivation. Second, we examine the role of the linker between the inactivation segment and TM1. The results show that the critical determinant for inactivation is an initial segment of three amino acids (residues 2-4: FIW) after the initiation methionine. Deletions that scan positions from residue 5 through residue 36 alter inactivation, but do not abolish it. In contrast, deletion of FIW or combinations of point mutations within the FIW triplet abolish inactivation. Mutational analysis of the three initial residues argues that inactivation does not result from a well-defined structure formed by this epitope. Inactivation may be better explained by linear entry of the NH(2)-terminal peptide segment into the permeation pathway with residue hydrophobicity and size influencing the onset and recovery from inactivation. Examination of the ability of artificial, polymeric linkers to support inactivation suggests that a variety of amino acid sequences can serve as adequate linkers as long as they contain a minimum of 12 residues between the first transmembrane segment and the FIW triplet. Thus, neither a specific distribution of charge on the linker nor a specific structure in the linker is required to support the inactivation process.