Barry D. Kyle

The regulation of BK channel activity by pre- and post-translational modifications

Large conductance, Ca2+-activated K+ (BK) channels represent an important pathway for the outward flux of K+ ions from the intracellular compartment in response to membrane depolarization, and/or an elevation in cytosolic free [Ca2+]. They are functionally expressed in a range of mammalian tissues (e.g., nerve and smooth muscles), where they can either enhance or dampen membrane excitability. The diversity of BK channel activity results from the considerable alternative mRNA splicing and post-translational modification (e.g., phosphorylation) of key domains within the pore-forming α subunit of the channel complex. Most of these modifications are regulated by distinct upstream cell signaling pathways that influence the structure and/or gating properties of the holo-channel and ultimately, cellular function. The channel complex may also contain auxiliary subunits that further affect channel gating and behavior, often in a tissue-specific manner. Recent studies in human and animal models have provided strong evidence that abnormal BK channel expression/function contributes to a range of pathologies in nerve and smooth muscle. By targeting the upstream regulatory events modulating BK channel behavior, it may be possible to therapeutically intervene and alter BK channel expression/function in a beneficial manner.

CaV3.2 Channels and the Induction of Negative Feedback in Cerebral Arteries

Rationale: T-type (CaV3.1/CaV3.2) Ca2+ channels are expressed in rat cerebral arterial smooth muscle. Although present, their functional significance remains uncertain with findings pointing to a variety of roles. Objective: This study tested whether CaV3.2 channels mediate a negative feedback response by triggering Ca2+ sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca2+-activated K+ channels. Methods and Results: Micromolar Ni2+, an agent that selectively blocks CaV3.2 but not CaV1.2/CaV3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in CaV3.2−/− arteries. Structural analysis using 3-dimensional tomography, immunolabeling, and a proximity ligation assay next revealed the existence of microdomains in cerebral arterial smooth muscle which comprised sarcoplasmic reticulum and caveolae. Within these discrete structures, CaV3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca2+ influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca2+-induced Ca2+ release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca2+ imaging and perforated patch clamp electrophysiology demonstrated that Ni2+ suppressed Ca2+ sparks and consequently spontaneous transient outward K+ currents, large-conductance Ca2+-activated K+ channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca2+-activated K+ channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni2+. Key experiments on human cerebral arteries indicate that CaV3.2 is present and drives a comparable response to moderate constriction. Conclusions: These findings indicate for the first time that CaV3.2 channels localize to discrete microdomains and drive ryanodine receptor–mediated Ca2+ sparks, enabling large-conductance Ca2+-activated K+ channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.

Specific phosphorylation sites underlie the stimulation of a large conductance, Ca2+-activated K+ channel by cGMP-dependent protein kinase

Smooth muscle contractility and neuronal excitability are regulated by large conductance, Ca2+‐activated K+ (BKCa) channels, the activity of which can be increased after modulation by type I cGMP‐dependent protein kinase (cGKI) via nitric oxide (NO)/cGMP signaling. Our study focused on identifying key phosphorylation sites within the BKCa channel underlying functional enhancement of channel activity by cGKI. BKCa channel phosphorylation by cGKIα was characterized biochemically using radiolabeled ATP, and regulation of channel activity by NO/cGMP signaling was quantified in rat aortic A7r5 smooth muscle cells by cell‐attached patch‐clamp recording. Serine to alanine substitutions at 3 of 6 putative cGKI phosphorylation sites (Ser691, Ser873, and Ser1112) in the BKCa α subunit individually reduced direct channel phosphorylation by 25–60% and blocked BKCa activation by either an NO donor or a membrane‐permeable cGMP by 80–100%. Acute inhibition of cGKI prevented stimulus‐evoked enhancement of BKCa channel activity. Our data further suggest that augmentation of BKCa activity by NO/cGMP/cGKI signaling requires phosphorylation at all 3 sites and is independent of elevations in [Ca2+]i. Phosphorylation of 3 specific Ser residues within the murine BKCa α subunit by cGKIα accounts for the enhanced BKCa channel activity induced by elevated [cGMP]i in situ.—Kyle, B.D., Hurst, S., Swayze, R. D., Sheng, J., Braun, A.P. Specific phosphorylation sites underlie the stimulation of a large conductance, Ca2+‐activated K+ channel by cGMP‐dependent protein kinase. FASEB J. 27, 2027–2038 (2013). www.fasebj.org

Cysteine string protein limits expression of the large conductance, calcium-activated K⁺ (BK) channel.

Large-conductance, calcium-activated K+ (BK) channels are widely distributed throughout the nervous system and play an essential role in regulation of action potential duration and firing frequency, along with neurotransmitter release at the presynaptic terminal. We have previously demonstrated that select mutations in cysteine string protein (CSPα), a presynaptic J-protein and co-chaperone, increase BK channel expression. This observation raised the possibility that wild-type CSPα normally functions to limit neuronal BK channel expression. Here we show by Western blot analysis of transfected neuroblastoma cells that when BK channels are present at elevated levels, CSPα acts to reduce expression. Moreover, we demonstrate that the accessory subunits, BKβ4 and BKβ1 do not alter CSPα-mediated reduction of expressed BKα subunits. Structure-function analysis reveals that the N-terminal J-domain of CSPα is critical for the observed regulation of BK channels levels. Finally, we demonstrate that CSPα limits BK current amplitude, while the loss-of-function homologue CSPαHPD-AAA increases BK current. Our observations indicate that CSPα has a role in regulating synaptic excitability and neurotransmission by limiting expression of BK channels.

The Large Conductance, Calcium-activated K + (BK) Channel is regulated by Cysteine String Protein

Large-conductance, calcium-activated-K+ (BK) channels are widely distributed throughout the nervous system, where they regulate action potential duration and firing frequency, along with presynaptic neurotransmitter release. Our recent efforts to identify chaperones that target neuronal ion channels have revealed cysteine string protein (CSPα) as a key regulator of BK channel expression and current density. CSPα is a vesicle-associated protein and mutations in CSPα cause the hereditary neurodegenerative disorder, adult-onset autosomal dominant neuronal ceroid lipofuscinosis (ANCL). CSPα null mice show 2.5 fold higher BK channel expression compared to wild type mice, which is not seen with other neuronal channels (i.e. Cav2.2, Kv1.1 and Kv1.2). Furthermore, mutations in either CSPαs J domain or cysteine string region markedly increase BK expression and current amplitude. We conclude that CSPα acts to regulate BK channel expression, and consequently CSPα-associated changes in BK activity may contribute to the pathogenesis of neurodegenerative disorders, such as ANCL.

Regional Variation in Arterial Myogenic Responsiveness: Links to Potassium Channel Diversity/Function

Regional variation in small artery myogenic responsiveness is associated with differences in relationships amongst intraluminal pressure, smooth muscle cell (SMC) membrane potential (Em) and vessel diameter. For example, under in vitro conditions, small arteries from cremaster muscle show a steeper relationship between Em and myogenic contraction compared with cerebral arteries. To explain this difference, we hypothesized that the function/regulation of the large conductance, Ca2+-activated, K+ channel (BKCa) differs between these vascular beds. This was based on previous observations by Nelson and colleagues that BKCa, activated by sarcoplasmic reticulum (SR)-generated Ca2+ sparks, exerts a hyperpolarizing influence that opposes myogenic constriction. To test this, studies were performed using Ca2+ imaging, vessel myography, isolated cell electrophysiology and molecular biology techniques on small resistance arteries from the cerebral and cremaster muscle vasculatures. While BKCa in SMCs of both small arteries showed a similar conductance and voltage sensitivity, Ca2+ sensitivity was 2–3-fold greater in cerebral SMCs. Single channel open times were greater in cerebral SMCs compared with those of cremaster SMCs. Conversely, closed times were significantly shorter in cerebral SMCs. In addition to variation in biophysical characteristics, β1-BKCa subunit expression was decreased in cremaster SMCs. Further, siRNA-induced knockdown of the β1 subunit of the BKCa holo-channel shifted gating behavior of cerebral BKCa channels to resemble that observed in cremaster SMCs. Collectively, the data indicate that while BKCa is present in both vascular preparations expression levels and modes of regulation differ. In particular, BKCa in small cerebral arteries is configured to show a higher Ca2+ sensitivity resulting in greater opening at physiological levels of membrane potential. Heterogeneity in SMC ion channel function is not limited to BKCa as vascular bed differences are also apparent for other K+ channels including the voltage-gated Kv and Kv7 families of channels. With respect to the latter, Kv7 channels appear to play a greater role in cerebral vasculature compared to the coronary circulation. From a physiological perspective it is suggested that differences in local ion channel function allow for regional differences in the regulation of myogenic tone and hence the control of tissue hemodynamics.

Correction: Cysteine String Protein Limits Expression of the Large Conductance, Calcium-Activated K+ (BK) Channel.

Fig 1C is incorrect as it shows the wrong actin blot. The authors have provided a corrected version of Fig 1 here. Fig 1 CSPα alters BK channel expression. Additionally, there is a sentence missing from the caption for Fig 3. Please see the complete, correct Fig 3 caption here. The missing sentence is highlighted in bold. Fig 3 The J domain of CSPα reduces BK channel expression.