Christopher J. Lingle

Multiple regulatory sites in large-conductance calcium-activated potassium channels

Large conductance, Ca2+- and voltage-activated K+ channels (BK) respond to two distinct physiological signals—membrane voltage and cytosolic Ca2+ (refs 1, 2). Channel opening is regulated by changes in Ca2+ concentration spanning 0.5 µM to 50 mM (refs 2–5), a range of Ca2+ sensitivity unusual among Ca2+-regulated proteins. Although voltage regulation arises from mechanisms shared with other voltage-gated channels, the mechanisms of Ca2+ regulation remain largely unknown. One potential Ca2+-regulatory site, termed the ‘Ca2+ bowl’, has been located to the large cytosolic carboxy terminus. Here we show that a second region of the C terminus, the RCK domain (regulator of conductance for K+ (ref. 12)), contains residues that define two additional regulatory effects of divalent cations. One site, together with the Ca2+ bowl, accounts for all physiological regulation of BK channels by Ca2+; the other site contributes to effects of millimolar divalent cations that may mediate physiological regulation by cytosolic Mg2+ (refs 5, 13). Independent regulation by multiple sites explains the large concentration range over which BK channels are regulated by Ca2+. This allows BK channels to serve a variety of physiological roles contingent on the Ca2+ concentration to which the channels are exposed.

Calcium sensitivity of BK-type KCa channels determined by a separable domain

High conductance, Ca(2+)-activated (BK-type) K+ channels from mouse (mSlo) and Drosophila (dSlo) differ in their functional properties but share a conserved core resembling voltage-gated K+ channels and a tail appended to the core by a nonconserved linker. We have found that the channel subunit is physically divisible into these two conserved domains and that the core determines such properties as channel open time, conductance, and, probably, voltage dependence, whereas the tail determines apparent Ca2+ sensitivity. Both domains are required for function. We demonstrated the different roles of the core and tail by taking advantage of the functional differences between mSlo and dSlo. Heterologous pairing of cores and tails from mSlo and dSlo showed that single-channel properties were always characteristic of the core species, but that apparent Ca2+ sensitivity was adjusted up or down depending on the species of the tail. Thus, the tail is implicated in the Ca(2+)-sensing role of BK channels.

Divalent cation sensitivity of BK channel activation supports the existence of three distinct binding sites

Mutational analyses have suggested that BK channels are regulated by three distinct divalent cation-dependent regulatory mechanisms arising from the cytosolic COOH terminus of the pore-forming α subunit. Two mechanisms account for physiological regulation of BK channels by μM Ca2+. The third may mediate physiological regulation by mM Mg2+. Mutation of five aspartate residues (5D5N) within the so-called Ca2+ bowl removes a portion of a higher affinity Ca2+ dependence, while mutation of D362A/D367A in the first RCK domain also removes some higher affinity Ca2+ dependence. Together, 5D5N and D362A/D367A remove all effects of Ca2+ up through 1 mM while E399A removes a portion of low affinity regulation by Ca2+/Mg2+. If each proposed regulatory effect involves a distinct divalent cation binding site, the divalent cation selectivity of the actual site that defines each mechanism might differ. By examination of the ability of various divalent cations to activate currents in constructs with mutationally altered regulatory mechanisms, here we show that each putative regulatory mechanism exhibits a unique sensitivity to divalent cations. Regulation mediated by the Ca2+ bowl can be activated by Ca2+ and Sr2+, while regulation defined by D362/D367 can be activated by Ca2+, Sr2+, and Cd2+. Mn2+, Co2+, and Ni2+ produce little observable effect through the high affinity regulatory mechanisms, while all six divalent cations enhance activation through the low affinity mechanism defined by residue E399. Furthermore, each type of mutation affects kinetic properties of BK channels in distinct ways. The Ca2+ bowl mainly accelerates activation of BK channels at low [Ca2+], while the D362/D367-related high affinity site influences both activation and deactivation over the range of 10–300 μM Ca2+. The major kinetic effect of the E399-related low affinity mechanism is to slow deactivation at mM Mg2+ or Ca2+. The results support the view that three distinct divalent-cation binding sites mediate regulation of BK channels.

Deletion of the Slo3 gene abolishes alkalization-activated K+ current in mouse spermatozoa

Mouse spermatozoa express a pH-dependent K+ current (KSper) thought to be composed of subunits encoded by the Slo3 gene. However, the equivalence of KSper and Slo3-dependent current remains uncertain, because heterologous expression of Slo3 results in currents that are less effectively activated by alkalization than are native KSper currents. Here, we show that genetic deletion of Slo3 abolishes all pH-dependent K+ current at physiological membrane potentials in corpus epididymal sperm. A residual pH-dependent outward current (IKres) is observed in Slo3−/− sperm at potentials of >0 mV. Differential inhibition of KSper/Slo3 and IKres by clofilium reveals that the amplitude of IKres is similar in both wild-type (wt) and Slo3−/− sperm. The properties of IKres suggest that it likely represents outward monovalent cation flux through CatSper channels. Thus, KSper/Slo3 may account for essentially all mouse sperm K+ current and is the sole pH-dependent K+ conductance in these sperm. With physiological ionic gradients, alkalization depolarizes Slo3−/− spermatozoa, presumably from CatSper activation, in contrast to Slo3/KSper-mediated hyperpolarization in wt sperm. Slo3−/− male mice are infertile, but Slo3−/− sperm exhibit some fertility within in vitro fertilization assays. Slo3−/− sperm exhibit a higher incidence of morphological abnormalities accentuated by hypotonic challenge and also exhibit deficits in motility in the absence of bicarbonate, revealing a role of KSper under unstimulated conditions. Together, these results show that KSper/Slo3 is the primary spermatozoan K+ current, that KSper may play a critical role in acquisition of normal morphology and sperm motility when faced with hyperosmotic challenges, and that Slo3 is critical for fertility.

Halothane inhibits two components of calcium current in clonal (GH3) pituitary cells

The effect of halothane on isolated calcium (Ca2+) current of clonal (GH3) pituitary cells was investigated using standard whole-cell clamp techniques at room temperature. Halothane (0.1–5.0 mM) reversibly reduced both the low-threshold, transient [low-voltage-activated (LVA)] component and the high-threshold [high-voltage-activated (HVA)] component of Ca2+ current. Halothane had little effect on the voltage dependence of activation or inactivation of either component of Ca2+ current. Inhibition of the peak high-threshold Ca2+ current was half- maximal at about 0.8 mM halothane, with maximal inhibition (100%) occurring with 5 mM halothane. When measured at the end of a 190-msec command step, half-maximal reduction of high-threshold current occurred at less than 0.5 mM halothane. The low-threshold transient current was less sensitive to halothane, with half-maximal inhibition of peak transient current activated at -30 mV occurring at approximately 1.3 mM. The effect of halothane on the HVA current was apparently not mediated by changes in intracellular Ca2+ concentration. The ability of halothane to inhibit Ca2+ current was unaffected by either the inclusion of the rapid Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid (BAPTA) in the recording pipette or exposure of the cell to 10 mM caffeine. To assess the selectivity of the effect of halothane, the actions of halothane on two components of voltage- activated potassium (K+) current observed in the absence of extracellular Ca2+ and on voltage-dependent sodium (Na+) current were also examined. Halothane had no effect on the voltage-dependent, inactivating K+ current of GH3 cells at concentrations up to 1.2 mM. In contrast, the non-inactivating K+ current, though less sensitive to halothane than either Ca2+ current, was reduced by about 40% by 1.2 mM halothane at +20 mV. Peak Na+ current was also blocked by halothane, but 50% block required around 2.6 mM halothane with little effect at 1.6 mM. Reduction of Na+ current was associated with a substantial negative shift in the steady-state inactivation curve. Although the results indicate that a number of voltage-dependent ionic currents are sensitive to halothane, both components of Ca2+ current exhibit a greater sensitivity to halothane than any of three other voltage- dependent currents in GH3 cells. These results show that GH3 cell Ca2+ currents are selectively inhibited by clinically appropriate concentrations of halothane and that the reduction of Ca2+ current can account for the inhibition by halothane of TRH- or KCl-induced prolactin secretion in GH3 cells.

Two components of calcium‐activated potassium current in rat adrenal chromaffin cells.

1. The activation of calcium (Ca2+)‐dependent potassium (K+) currents in dissociated rat adrenal chromaffin cells was investigated using the dialysed cell recording technique. 2. Ca(2+)‐dependent K+ current was the major component of outward current at command potentials from ‐30 mV to about +50 mV. 3. Two components of Ca(2+)‐dependent outward current could be distinguished based on the voltage dependence of activation, the properties of tail currents following repolarization, and pharmacological properties. 4. One Ca(2+)‐dependent current was similar to an after‐hyperpolarization current (often termed IAHP) observed in other cell types. This current was largely blocked by 200 nM‐apamin or 200 microM‐curare, was associated with slow Ca(2+)‐dependent tail current, and exhibited little dependence on voltage. In cells with cytosolic Ca2+ buffered to 500 nM‐1 microM, curare‐sensitive current accounted for most of the membrane current at potentials negative to about ‐40 mV. 5. A second component of Ca(2+)‐activated K+ current exhibited voltage‐dependent activation, was completely blocked by 1 mM‐TEA, and turned off rapidly following repolarization. An unusual aspect of the TEA‐sensitive currents was that they appeared to inactivate under conditions of constant cytosolic Ca2+. 6. A novel observation during these experiments was a slow hump of outward current which appears to result from a non‐monotonic elevation in cytosolic Ca2+ during prolonged voltage jumps.

Redox-sensitive extracellular gates formed by auxiliary β 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 β3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming α subunits. BK β subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the β3 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 β subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the α subunits to influence or block access of small molecules to the permeation pathway.

Calcium-Activated Potassium Channels in Adrenal Chromaffin Cells

Rat chromaffin cells express an interesting diversity of Ca(2+)-dependent K+ channels, including a voltage-independent, small-conductance, apamin-sensitive SK channel and two variants of voltage-dependent, large-conductance BK channels. The two BK channel variants are differentially segregated among chromaffin cells, such that BK current is completely inactivating in about 75-80% of rat chromaffin cells, while the remainder express a mix of inactivating and non-inactivating current or mostly non-inactivating BKs current. The single-channel conductance of BKi channels is identical to that of BKs channels. Although rates of current activation are similar in the two variants, the deactivation kinetics of the two channels also differ. Furthermore, BKi channels are somewhat less sensitive to scorpion toxins than BKs channels. The slow component of BKi channel deactivation may be an important determinant of the functional role of these channels. During blockade of SK current, cells with BKi current fire tonically during sustained depolarizing current injection, whereas cells with BKs current tend to fire only a few action potentials before becoming quiescent. The ability to repetitively fire requires functional BKi channels, since partial blockade of BKi channels by CTX makes a BKi cell behave much like a BKs cell. In contrast, the physiological significance of BKi inactivation may arise from the ability of secretagogue-induced [Ca2+]i elevations to regulate the availability of BKi channels during subsequent action potentials (Herrington et al., 1995). By reducing the number of BK channels available for repolarization, the time course of action potentials may be prolonged. This possibility remains to be tested directly. These results raise a number of interesting questions pertinent to the control of secretion in rat adrenal chromaffin cells. An interesting hypothesis is that cells with a particular kind of BK current may reflect particular subpopulations of chromaffin cells. These subpopulations might differ either in the nature of the material secreted from the cell (e.g., Douglass and Poisner, 1965) or in the responsiveness to particular secretagogues. The differences in electrical behavior between cells with BKi and BKs current suggest that the pattern of secretion that might be elicited by a single type of stimulus could differ. For BKi cells, secretion may occur in a tonic fashion during sustained depolarization, while secretion from cells with BKs current may be more phasic. In the absence of specific structural information about the domains responsible for inactivation of BKi channels, our understanding of the mechanism of inactivation remains indirect. BKi inactivation shares many features with N-terminal inactivation of voltage-dependent K+ channels. However, there are provocative differences between the two types of inactivation which require us to propose that the native inactivation domain of BKi channels may occlude access of permeant ions to the BK channel permeation pathway in a position at some distance from the actual mouth of the channel. Further understanding of the structural and mechanistic basis of inactivation of BKi channels promises to provide new insights into both the cytoplasmic topology of BK channels and the Ca(2+)- and voltage-dependent steps involved in channel activation.

Inactivating BK Channels in Rat Chromaffin Cells May Arise from Heteromultimeric Assembly of Distinct Inactivation-Competent and Noninactivating Subunits

Inactivating and noninactivating variants of large-conductance, Ca2+-dependent, voltage-dependent BK-type channels are found in rat chromaffin cells and are largely segregated into different cells. Here we test the hypothesis that, within the population of cells that express inactivating BK current (BKi current), the BKi channels are largely heteromultimers composed of inactivation-competent subunits (bk(i)) and noninactivating subunits (bk(s)). Several independent types of evidence support this view. The gradual removal of inactivation by trypsin is consistent with the idea that in most cells and patches there are, on average, about two to three inactivation domains per channel. In addition, several aspects of blockade of BKi current by charybdotoxin (CTX) are consistent with the idea that BKi channels contain differing numbers (one to four) of relatively CTX-resistant bk(i) subunits. Finally, the frequency of occurrence of noninactivating BKs channels in patches with predominantly inactivating BKi channels is consistent with the binomial expectations of random, independent assembly of two distinct subunits, if most cells have, on average, about two to three bk(i) subunits per channel. These results suggest that the phenotypic properties of BKi currents and the resulting cellular electrical excitability may exhibit a continuum of behavior that arises simply from the differential expression of two distinct subunits.

Inactivation of BK Channels by the NH2 Terminus of the β2 Auxiliary Subunit: An Essential Role of a Terminal Peptide Segment of Three Hydrophobic Residues

An auxiliary β2 subunit, when coexpressed with Slo α subunits, produces inactivation of the resulting large-conductance, Ca2+ and voltage-dependent K+ (BK-type) channels. Inactivation is mediated by the cytosolic NH2 terminus of the β2 subunit. To understand the structural requirements for inactivation, we have done a mutational analysis of the role of the NH2 terminus in the inactivation process. The β2 NH2 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 NH2-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.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.