Richard W. Aldrich

Coupling between Voltage Sensor Activation, Ca2+ Binding and Channel Opening in Large Conductance (BK) Potassium Channels

To determine how intracellular Ca2+ and membrane voltage regulate the gating of large conductance Ca2+-activated K+ (BK) channels, we examined the steady-state and kinetic properties of mSlo1 ionic and gating currents in the presence and absence of Ca2+ over a wide range of voltage. The activation of unliganded mSlo1 channels can be accounted for by allosteric coupling between voltage sensor activation and the closed (C) to open (O) conformational change (Horrigan, F.T., and R.W. Aldrich. 1999. J. Gen. Physiol. 114:305–336; Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277–304). In 0 Ca2+, the steady-state gating charge-voltage (QSS-V) relationship is shallower and shifted to more negative voltages than the conductance-voltage (GK-V) relationship. Calcium alters the relationship between Q-V and G-V, shifting both to more negative voltages such that they almost superimpose in 70 μM Ca2+. This change reflects a differential effect of Ca2+ on voltage sensor activation and channel opening. Ca2+ has only a small effect on the fast component of ON gating current, indicating that Ca2+ binding has little effect on voltage sensor activation when channels are closed. In contrast, open probability measured at very negative voltages (less than −80 mV) increases more than 1,000-fold in 70 μM Ca2+, demonstrating that Ca2+ increases the C-O equilibrium constant under conditions where voltage sensors are not activated. Thus, Ca2+ binding and voltage sensor activation act almost independently, to enhance channel opening. This dual-allosteric mechanism can reproduce the steady-state behavior of mSlo1 over a wide range of conditions, with the assumption that activation of individual Ca2+ sensors or voltage sensors additively affect the energy of the C-O transition and that a weak interaction between Ca2+ sensors and voltage sensors occurs independent of channel opening. By contrast, macroscopic IK kinetics indicate that Ca2+ and voltage dependencies of C-O transition rates are complex, leading us to propose that the C-O conformational change may be described by a complex energy landscape.

Astrocytic endfoot Ca2+ and BK channels determine both arteriolar dilation and constriction

Neuronal activity is thought to communicate to arterioles in the brain through astrocytic calcium (Ca2+) signaling to cause local vasodilation. Paradoxically, this communication may cause vasoconstriction in some cases. Here, we show that, regardless of the mechanism by which astrocytic endfoot Ca2+ was elevated, modest increases in Ca2+ induced dilation, whereas larger increases switched dilation to constriction. Large-conductance, Ca2+-sensitive potassium channels in astrocytic endfeet mediated a majority of the dilation and the entire vasoconstriction, implicating local extracellular K+ as a vasoactive signal for both dilation and constriction. These results provide evidence for a unifying mechanism that explains the nature and apparent duality of the vascular response, showing that the degree and polarity of neurovascular coupling depends on astrocytic endfoot Ca2+ and perivascular K+.

LRRC26 auxiliary protein allows BK channel activation at resting voltage without calcium

Large-conductance, voltage- and calcium-activated potassium (BK, or KCa1.1) channels are ubiquitously expressed in electrically excitable and non-excitable cells, either as α-subunit (BKα) tetramers or together with tissue specific auxiliary β-subunits (β1–β4). Activation of BK channels typically requires coincident membrane depolarization and elevation in free cytosolic Ca2+ concentration ([Ca2+]i), which are not physiological conditions for most non-excitable cells. Here we present evidence that in non-excitable LNCaP prostate cancer cells, BK channels can be activated at negative voltages without rises in [Ca2+]i through their complex with an auxiliary protein, leucine-rich repeat (LRR)-containing protein 26 (LRRC26). LRRC26 modulates the gating of a BK channel by enhancing the allosteric coupling between voltage-sensor activation and the channel’s closed–open transition. This finding reveals a novel auxiliary protein of a voltage-gated ion channel that gives an unprecedentedly large negative shift (∼−140 mV) in voltage dependence and provides a molecular basis for activation of BK channels at physiological voltages and calcium levels in non-excitable cells.

Allosteric linkage between voltage and Ca(2+)-dependent activation of BK-type mslo1 K(+) channels.

The activation of BK type Ca(2+)-activated K(+) channels depends on both voltage and Ca(2+). We studied three point mutations in the putative voltage sensor S4 or S4-S5 linker regions in the mslo1 BK channels to explore the relationship between voltage and Ca(2+) in activating the channel. These mutations reduced the steepness of the open probability - voltage (P(o) - V) relation and increased the shift of the P(o) - V relations on the voltage axis in response to increases in the calcium concentration. It is striking that these two effects were reciprocally related for all three mutations, despite different effects of the mutations on other aspects of the voltage dependence of channel gating. This reciprocal relationship suggests strongly that the free energy contributions to channel activation provided by voltage and by calcium binding are simply additive. We conclude that the Ca(2+) binding sites and the voltage sensors do not directly interact. Rather they both affect the mslo1 channel opening through an allosteric mechanism, by influencing the conformational change between the closed and open conformations. The mutations changed the channels voltage dependence with little effect on its Ca(2+) affinitiy.

BK potassium channel modulation by leucine-rich repeat-containing proteins

Molecular diversity of ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and nonexcitable cells. Regulation by variable auxiliary subunits is a major mechanism to generate tissue- or cell-specific diversity of ion channel function. Mammalian large-conductance, voltage- and calcium-activated potassium channels (BK, KCa1.1) are ubiquitously expressed with diverse functions in different tissues or cell types, consisting of the pore-forming, voltage- and Ca2+-sensing α-subunits (BKα), either alone or together with the tissue-specific auxiliary β-subunits (β1–β4). We recently identified a leucine-rich repeat (LRR)-containing membrane protein, LRRC26, as a BK channel auxiliary subunit, which causes an unprecedented large negative shift (∼140 mV) in voltage dependence of channel activation. Here we report a group of LRRC26 paralogous proteins, LRRC52, LRRC55, and LRRC38 that potentially function as LRRC26-type auxiliary subunits of BK channels. LRRC52, LRRC55, and LRRC38 produce a marked shift in the BK channel’s voltage dependence of activation in the hyperpolarizing direction by ∼100 mV, 50 mV, and 20 mV, respectively, in the absence of calcium. They along with LRRC26 show distinct expression in different human tissues: LRRC26 and LRRC38 mainly in secretory glands, LRRC52 in testis, and LRRC55 in brain. LRRC26 and its paralogs are structurally and functionally distinct from the β-subunits and we designate them as a γ family of the BK channel auxiliary proteins, which potentially regulate the channel’s gating properties over a spectrum of different tissues or cell types.

Erectile dysfunction in mice lacking in large conductance calcium-activated (BK) channel

Penile erection is dependent on the nitric oxide (NO)/cGMP‐dependent protein kinase I (PKGI) pathway. One important target of PKGI in smooth muscle is the large‐conductance, calcium‐activated potassium (BK) channel, which upon activation hyperpolarizes the smooth muscle cell membrane, causing relaxation. Relaxation of arterial and corpus cavernosum smooth muscle (CCSM) is necessary to increase blood flow into the corpora cavernosa that leads to penile tumescence. We investigated the functional role of BK channels in the corpus cavernosum utilizing a knock‐out mouse lacking the Slo gene (Slo−/−) responsible for the pore‐forming subunit of the BK channel. Whole‐cell currents were recorded from isolated CCSM cells of Slo+/+ and Slo−/− mice. Iberiotoxin‐sensitive voltage‐ and [Ca2+]‐activated K+ currents, the latter activated by local transient calcium releases (calcium sparks), were present in Slo+/+ CCSM cells, but absent in Slo−/− cells. CCSM strips from Slo−/− mice demonstrated a four‐fold increase in phasic contractions, in the presence of phenylephrine. Nerve‐evoked relaxations of precontracted strips were reduced by 50%, both in strips from Slo−/− mice and by blocking BK channels with iberiotoxin in the Slo+/+ strips. Consistent with the in vitro results, in vivo intracavernous pressure exhibited pronounced oscillations in Slo−/− mice, but not in Slo+/+ mice. Furthermore, intracavernous pressure increases to nerve stimulation, in vivo, were reduced by 22% in Slo−/−mice. These results indicate that the BK channel has an important role in erectile function, and loss of the BK channel leads to erectile dysfunction.

Profiling the Phospho-status of the BKCa Channel α Subunit in Rat Brain Reveals Unexpected Patterns and Complexity

Molecular diversity of ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and non-excitable cells. Protein phosphorylation and alternative splicing of pre-mRNA are two important mechanisms to generate structural and functional diversity of ion channels. However, systematic mass spectrometric analyses of in vivo phosphorylation and splice variants of ion channels in native tissues are largely lacking. Mammalian large-conductance calcium-activated potassium (BKCa) channels are tetramers of α subunits (BKα) either alone or together with β subunits, exhibit exceptionally large single channel conductance, and are dually activated by membrane depolarization and intracellular Ca2+. The cytoplasmic C terminus of BKα is subjected to extensive pre-mRNA splicing and, as predicted by several algorithms, offers numerous phospho-acceptor amino acids. Here we use nanoflow liquid chromatography tandem mass spectrometry on BKCa channels affinity-purified from rat brain to analyze in vivo BKα phosphorylation and splicing. We found 7 splice variations and identified as many as 30 Ser/Thr in vivo phosphorylation sites; most of which were not predicted by commonly used algorithms. Of the identified phosphosites 23 are located in the C terminus, four were found on splice insertions. Electrophysiological analyses of phospho- and dephosphomimetic mutants transiently expressed in HEK-293 cells suggest that phosphorylation of BKα differentially modulates the voltage- and Ca2+-dependence of channel activation. These results demonstrate that the pore-forming subunit of BKCa channels is extensively phosphorylated in the mammalian brain providing a molecular basis for the regulation of firing pattern and excitability through dynamic modification of BKα structure and function.

State-independent Block of BK Channels by an Intracellular Quaternary Ammonium

Intracellular blockade by quaternary ammonium (QA) molecules of many potassium channels is state dependent, where the requirement for channel opening is evidenced by a time-dependent component of block in the macroscopic record. Whether this is the case for Ca2+- and voltage-activated potassium (BK) channels, however, remains unclear. Previous work (Li, W., and R.W. Aldrich. 2004. J. Gen. Physiol. 124:43–57) tentatively proposed a state-dependent, trapping model, but left open the possibility of state-independent block. Here, we found BK channel blockade by a novel QA derivative, bbTBA, was time dependent, raising the possibility of state-dependent, open channel block. Alternatively, the observed voltage dependence of block could be sufficient to explain time-dependent block. We have used steady-state and kinetic measurements of bbTBA blockade in order to discriminate between these two possibilities. bbTBA did not significantly slow deactivation kinetics at potentials between −200 and −100 mV, suggesting that channels can close unhindered by bound bbTBA. We further find no evidence that bbTBA is trapped inside BK channels after closing. Measurements of steady state fractional block at +40 mV revealed a 1.3-fold change in apparent affinity for a 33-fold change in Po, in striking contrast to the 31-fold change predicted by state-dependent block. Finally, the appearance of a third kinetic component of bbTBA blockade at high concentrations is incompatible with state-dependent block. Our results suggest that access of intracellular bbTBA to the BK channel cavity is not strictly gated by channel opening and closing, and imply that the permeation gate for BK channels may not be intracellular.

Calcium activation of BK(Ca) potassium channels lacking the calcium bowl and RCK domains.

In many physiological systems such as neurotransmitter release, smooth muscle relaxation and frequency tuning of auditory hair cells, large-conductance calcium-activated potassium (BKCa) channels create a connection between calcium signalling pathways and membrane excitability. BKCa channels are activated by voltage and by micromolar concentrations of intracellular calcium. Although it is possible to open BKCa channels in the absence of calcium, calcium binding is essential for their activation under physiological conditions. In the presence of intracellular calcium, BKCa channels open at more negative membrane potentials. Many experiments investigating the molecular mechanism of calcium activation of the BKCa channel have focused on the large intracellular carboxy terminus, and much evidence supports the hypothesis that calcium-binding sites are located in this region of the channel. Here we show that BKCa channels that lack the whole intracellular C terminus retain wild-type calcium sensitivity. These results show that the intracellular C terminus, including the ‘calcium bowl’ and the RCK domain, is not necessary for the calcium-activated opening of these channels.

Cochlear Function in Mice Lacking the BK Channel α, β1, or β4 Subunits

Large conductance voltage- and calcium-activated potassium (BK) channels are important for regulating many essential cellular functions, from neuronal action potential shape and firing rate to smooth muscle contractility. In amphibians, reptiles, and birds, BK channels mediate the intrinsic frequency tuning of the cochlear hair cell by an electrical resonance mechanism. In contrast, inner hair cells of the mammalian cochlea are extrinsically tuned by accessory structures of the cochlea. Nevertheless, BK channels are present in inner hair cells and encode a fast activating outward current. To understand the role of the BK channel α and β subunits in mammalian inner hair cells, we analyzed the morphology, physiology, and function of these cells from mice lacking the BK channel α (Slo-/-) and also the β1 and β4 subunits (β1/4-/-). β1/4-/- mice showed normal subcellular localization, developmental acquisition, and expression of BK channels. β1/4-/- mice showed normal cochlear function as indicated by normal auditory brainstem responses and distortion product otoacoustic emissions. Slo-/- mice also showed normal cochlear function despite the absence of the BKα subunit and the absence of fast activating outward current from the inner hair cells. Moreover, microarray analyses revealed no compensatory changes in transcripts encoding ion channels or transporters in the cochlea from Slo-/- mice. Slo-/- mice did, however, show increased resistance to noise-induced hearing loss. These findings reveal the fundamentally different contribution of BK channels to nonmammalian and mammalian hearing and suggest that BK channels should be considered a target in the prevention of noise-induced hearing loss.