Xu-Hui Zeng

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.

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.

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.

LRRC52 (leucine-rich-repeat-containing protein 52), a testis-specific auxiliary subunit of the alkalization-activated Slo3 channel

KSper, a pH-dependent K+ current in mouse spermatozoa that is critical for fertility, is activated by alkalization in the range of pH 6.4–7.2 at membrane potentials between −50 and 0 mV. Although the KSper pore-forming subunit is encoded by the Slo3 gene, heterologously expressed Slo3 channels are largely closed at potentials negative to 0 mV at physiological pH. Here we identify a Slo3-associating protein, LRRC52 (leucine-rich repeat-containing 52), that shifts Slo3 gating into a range of voltages and pH values similar to that producing KSper current activation. Message for LRRC52, a homolog of the Slo1-modifying LRRC26 protein, is enriched in testis relative to other homologous LRRC subunits and is developmentally regulated in concert with that for Slo3. LRRC52 protein is detected only in testis. It is markedly diminished from Slo3−/− testis and completely absent from Slo3−/− sperm, indicating that LRRC52 expression is critically dependent on the presence of Slo3. We also examined the ability of other LRRC subunits homologous to LRRC26 and LRRC52 to modify Slo3 currents. Although both LRRC26 and LRRC52 are able to modify Slo3 function, LRRC52 is the stronger modifier of Slo3 function. Effects of other related subunits were weaker or absent. We propose that LRRC52 is a testis-enriched Slo3 auxiliary subunit that helps define the specific alkalization dependence of KSper activation. Together, LRRC52 and LRRC26 define a new family of auxiliary subunits capable of critically modifying the gating behavior of Slo family channels.

Slo3 K+ channels: Voltage and pH dependence of macroscopic currents

The mouse Slo3 gene (KCNMA3) encodes a K+ channel that is regulated by changes in cytosolic pH. Like Slo1 subunits responsible for the Ca2+ and voltage-activated BK-type channel, the Slo3 α subunit contains a pore module with homology to voltage-gated K+ channels and also an extensive cytosolic C terminus thought to be responsible for ligand dependence. For the Slo3 K+ channel, increases in cytosolic pH promote channel activation, but very little is known about many fundamental properties of Slo3 currents. Here we define the dependence of macroscopic conductance on voltage and pH and, in particular, examine Slo3 conductance activated at negative potentials. Using this information, the ability of a Horrigan-Aldrich–type of general allosteric model to account for Slo3 gating is examined. Finally, the pH and voltage dependence of Slo3 activation and deactivation kinetics is reported. The results indicate that Slo3 differs from Slo1 in several important ways. The limiting conductance activated at the most positive potentials exhibits a pH-dependent maximum, suggesting differences in the limiting open probability at different pH. Furthermore, over a 600 mV range of voltages (−300 to +300 mV), Slo3 conductance shifts only about two to three orders of magnitude, and the limiting conductance at negative potentials is relatively voltage independent compared to Slo1. Within the context of the Horrigan-Aldrich model, these results indicate that the intrinsic voltage dependence (zL) of the Slo3 closed–open equilibrium and the coupling (D) between voltage sensor movement are less than in Slo1. The kinetic behavior of Slo3 currents also differs markedly from Slo1. Both activation and deactivation are best described by two exponential components, both of which are only weakly voltage dependent. Qualitatively, the properties of the two kinetic components in the activation time course suggest that increases in pH increase the fraction of more rapidly opening channels.

Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa

During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca2+. The elevation of Ca2+ is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K+ current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca2+ current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1–4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K+ current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K+ current present in the Slo3 −/− sperm arise from CATSPER? Second, can any additional membrane K+ currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 −/− sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca2+ influx in response to alkalization.

Closed-channel block of BK potassium channels by bbTBA requires partial activation.

Blockade of large-conductance Ca2+-activated K+ (BK) channels by the bulky quaternary ammonium compound, N-(4-[benzoyl]benzyl)-N,N,N-tributylammonium (bbTBA), exhibits features consistent with blockade of both closed and open states. Here, we examine block of closed BK channels by bbTBA and how it may differ from block of open channels. Although our observations generally confirm earlier results, we describe three observations that are inconsistent with a model in which closed and open channels are equally accessible to blockade by bbTBA. First, block by bbTBA exhibits Ca2+-dependent features that are inconsistent with strictly state-independent block. Second, the steady-state voltage dependence of bbTBA block at negative potentials shows that any block of completely closed states either does not occur or is completely voltage independent. Third, determination of the fractional unblock by bbTBA at either low or high Ca2+ reveals deviations from a model in which open- and closed-state block is identical. The results support the view that bbTBA blockade of fully closed channels does not occur. We imagine two general types of explanation. First, a stronger voltage dependence of closed-channel block may minimize the contribution of closed-channel block at negative potentials. Second, voltage-dependent conformational changes among closed-channel states may permit block by bbTBA. The analysis supports the latter view, suggesting that bbTBA blockade of fully closed channels does not occur, but the ability of bbTBA to block a closed channel requires movement of one or more voltage sensors. Models in which block is coupled to voltage sensor movement can qualitatively account for (1) the ability of open-channel block to better fit block of conductance–voltage curves at high Ca2+; (2) the voltage dependence of fractional availability; and (3) the fractional unblock at different open probabilities. BK channels appear to undergo voltage-dependent conformational changes among closed states that are permissive for bbTBA block.

Differential Regulation of Action Potentials by Inactivating and Noninactivating BK Channels in Rat Adrenal Chromaffin Cells

Large-conductance Ca(2+)-activated K(+) (BK) channels can regulate cellular excitability in complex ways because they are able to respond independently to two distinct cellular signals, cytosolic Ca(2+) and membrane potential. In rat chromaffin cells (RCC), inactivating BK(i) and noninactivating (BK(s)) channels differentially contribute to RCC action potential (AP) firing behavior. However, the basis for these differential effects has not been fully established. Here, we have simulated RCC action potential behavior, using Markovian models of BK(i) and BK(s) current and other RCC currents. The analysis shows that BK current influences both fast hyperpolarization and afterhyperpolarization of single APs and that, consistent with experimental observations, BK(i) current facilitates repetitive firing of APs, whereas BK(s) current does not. However, the key functional difference between BK(i) and BK(s) current that accounts for the differential firing is not inactivation but the more negatively shifted activation range for BK(i) current at a given [Ca(2+)].

Interactions between β Subunits of the KCNMB Family and Slo3: β4 Selectively Modulates Slo3 Expression and Function

Background The pH and voltage-regulated Slo3 K+ channel, a homologue of the Ca2+- and voltage-regulated Slo1 K+ channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary β subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct β subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels. Methodology/Principal Findings To examine the ability of the KCNMB family of auxiliary β subunits to regulate Slo3 function, we co-expressed Slo3 and each β subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The β4 subunit produced an 8–10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither β1, β2, nor β3 mimicked the ability of β4 to increase surface expression, although biochemical tests suggested that all four β subunits are competent to coassemble with Slo3. Fluorescence microscopy from β4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that β4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that β4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm. Conclusions/Significance These results argue that, for native mouse Slo3 channels, the β4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 α subunit.