Andrew P. Braun

Enhanced activity of a large conductance, calcium-sensitive K+ channel in the presence of Src tyrosine kinase.

Large conductance, calcium-sensitive K+ channels (BKCa channels) contribute to the control of membrane potential in a variety of tissues, including smooth muscle, where they act as the target effector for intracellular “calcium sparks” and the endothelium-derived vasodilator nitric oxide. Various signal transduction pathways, including protein phosphorylation can regulate the activity of BKCa channels, along with many other membrane ion channels. In our study, we have examined the regulation of BKCa channels by the cellularSrc gene product (cSrc), a soluble tyrosine kinase that has been implicated in the regulation of both voltage- and ligand-gated ion channels. Using a heterologous expression system, we observed that co-expression of murine BKCa channel and the human cSrc tyrosine kinase in HEK 293 cells led to a calcium-sensitive enhancement of BKCa channel activity in excised membrane patches. In contrast, co-expression with a catalytically inactive cSrc mutant produced no change in BKCa channel activity, demonstrating the requirement for a functional cSrc molecule. Furthermore, we observed that BKCa channels underwent direct tyrosine phosphorylation in cells co-transfected with BKCa channels and active cSrc but not in cells co-transfected with the kinase inactive form of the enzyme. A single Tyr to Phe substitution in the C-terminal half of the channel largely prevented this observed phosphorylation. Given that cSrc may become activated by receptor tyrosine kinases or G-protein-coupled receptors, these findings suggest that cSrc-dependent tyrosine phosphorylation of BKCa channels in situ may represent a novel regulatory mechanism for altering membrane potential and calcium entry.

Large conductance, Ca2+‐activated K+ channels (BKCa) and arteriolar myogenic signaling

Myogenic, or pressure‐induced, vasoconstriction is critical for local blood flow autoregulation. Underlying this vascular smooth muscle (VSM) response are events including membrane depolarization, Ca2+ entry and mobilization, and activation of contractile proteins. Large conductance, Ca2+‐activated K+ channel (BKCa) has been implicated in several of these steps including, (1) channel closure causing membrane depolarization, and (2) channel opening causing hyperpolarization to oppose excessive pressure‐induced vasoconstriction. As multiple mechanisms regulate BKCa activity (subunit composition, membrane potential (Em) and Ca2+ levels, post‐translational modification) tissue level diversity is predicted. Importantly, heterogeneity in BKCa channel activity may contribute to tissue‐specific differences in regulation of myogenic vasoconstriction, allowing local hemodynamics to be matched to metabolic requirements. Knowledge of such variability will be important to exploiting the BKCa channel as a therapeutic target and understanding systemic effects of its pharmacological manipulation.

Integrin Receptor Activation Triggers Converging Regulation of Cav1.2 Calcium Channels by c-Src and Protein Kinase A Pathways

L-type, voltage-gated Ca2+ channels (CaL) play critical roles in brain and muscle cell excitability. Here we show that currents through heterologously expressed neuronal and smooth muscle CaL channel isoforms are acutely potentiated following α5β1 integrin activation. Only the α1C pore-forming channel subunit is critical for this process. Truncation and site-directed mutagenesis strategies reveal that regulation of Cav1.2 by α5β1 integrin requires phosphorylation of α1C C-terminal residues Ser1901 and Tyr2122. These sites are known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively, and are conserved between rat neuronal (Cav1.2c) and smooth muscle (Cav1.2b) isoforms. Kinase assays are consistent with phosphorylation of these two residues by PKA and c-Src. Following α5β1 integrin activation, native CaL channels in rat arteriolar smooth muscle exhibit potentiation that is completely blocked by combined PKA and Src inhibition. Our results demonstrate that integrin-ECM interactions are a common mechanism for the acute regulation of CaL channels in brain and muscle. These findings are consistent with the growing recognition of the importance of integrin-channel interactions in cellular responses to injury and the acute control of synaptic and blood vessel function.

Alpha 1-adrenoceptors reduce background K+ current in rabbit ventricular myocytes.

1. Ventricular myocytes were isolated by enzymatic dispersion of adult rabbit hearts, and voltage clamped using the whole‐cell variation of the patch clamp technique. Experiments were carried out at either 35 degrees C or room temperature (21‐23 degrees C). 2. In the presence of 10(‐3) M‐4‐aminopyridine to block the transient outward K+ current, and 10(‐6) M‐propranolol to block beta‐adrenoceptors, the alpha 1‐adrenergic agonist methoxamine produced action potential prolongation, and a small depolarization of the diastolic membrane potential. Under voltage clamp conditions, methoxamine decreased the magnitude of the inward rectifier K+ current, IK1, in both the inward and outward directions. This effect was dose dependent (10(‐5)‐10(‐3) M) and fully reversible upon wash‐out of the agonist. 3. The neurotransmitter noradrenaline (10(‐6)‐2 x 10(‐5) M), in the presence of propranolol (10(‐6) M), also reduced IK1 in ventricular cells, and this effect was blocked by the specific alpha 1‐adrenoceptor antagonist prazosin. 4. The alpha 1‐adrenoceptor‐mediated decrease in IK1 in ventricular myocytes was not affected by pre‐incubation of the cells with 0.5 micrograms/ml pertussis toxin (8‐10 h, 30‐32 degrees C). This result suggests that in rabbit ventricular cells, the alpha 1‐modulation of IK1 occurs via a pertussis toxin‐insensitive guanine nucleotide‐binding regulatory protein. 5. These observations demonstrate that IK1 in ventricular myocytes can be modulated by cardiac alpha 1‐adrenoceptors. The resulting changes in action potential repolarization and diastolic membrane potential may have significant effects on cardiac performance.

Openers of SKCa and IKCa channels enhance agonist-evoked endothelial nitric oxide synthesis and arteriolar vasodilation

Recent data have led us to hypothesize that selective activation of endothelial small‐ and/or intermediate‐conductance, calcium‐activated potassium channels (SKCa and IKCa channels, respectively) by the opener compounds NS309 and DCEBIO would augment stimulated nitric oxide (NO) synthesis and vasodilation in resistance arteries. Experimentally, ATP‐evoked changes in membrane potential, cytosolic Ca2+, and NO synthesis were recorded by patch clamp and microfluorimetry in single human endothelial cells. Agonist‐evoked inhibition of myogenic tone in isolated, pressurized arterioles from rat cremaster skeletal muscle was analyzed by video microscopy. NS309 and DCEBIO enhanced ATP‐evoked membrane hyperpolarization and cytosolic Ca2+ transients, along with acute NO synthesis in isolated endothelial cells. The acetylcholine‐mediated inhibition of myogenic tone (IC50 = 237 nM) was left‐shifted in the presence of NS309 and DCEBIO (10, 100, and 1000 nM) to IC50 values of 101, 78, and 43 nM;endothelial denudation inhibited this drug effect. l‐NAME attenuated the acetylcholine‐induced inhibition of myogenic tone but did not interfere with NS309 and DCEBIO‐evoked vasodilation. Collectively, our data demonstrate that drug‐induced enhancement of endothelial SKCa and IKCa channel activities represents a novel cellular mechanism to increase vasodilation of small‐resistance arterioles, thereby highlighting these channels as potential therapeutic targets in cardiovascular disease states associated with compromised NO signaling.—Sheng, J.‐S., Ella, S., Davis, M. J., Hill, M. A., Braun, A. P. Openers of SKCa and IKCa channels enhance agonist‐evoked endothelial nitric oxide synthesis and arteriolar vasodilation. FASEB J. 23, 1138–1145 (2009)

Heterogeneity in function of small artery smooth muscle BKCa: involvement of the β1‐subunit

Arteriolar myogenic vasoconstriction occurs when increased stretch or membrane tension leads to smooth muscle cell depolarization and opening of voltage‐gated Ca2+ channels. To prevent positive feedback and excessive pressure‐induced vasoconstriction, studies in cerebral artery smooth muscle have suggested that activation of large conductance, Ca2+‐activated K+ channels (BKCa) provides an opposing hyperpolarizing influence reducing Ca2+ channel activity. We have hypothesized that this mechanism may not equally apply to all vascular beds. To establish the existence of such heterogeneity in vascular reactivity, studies were performed on rat vascular smooth muscle (VSM) cells from cremaster muscle arterioles and cerebral arteries. Whole cell K+ currents were determined at pipette [Ca2+] of 100 nm or 5 μm in the presence and absence of the BKCa inhibitor, iberiotoxin (IBTX; 0.1 μm). Similar outward current densities were observed for the two cell preparations at the lower pipette Ca2+ levels. At 5 μm Ca2+, cremaster VSM showed a significantly (P < 0.05) lower current density compared to cerebral VSM (34.5 ± 1.9 vs 45.5 ± 1.7 pA pF−1 at +70 mV). Studies with IBTX suggested that the differences in K+ conductance at 5 μm intracellular [Ca2+] were largely due to activity of BKCa. 17β‐Oestradiol (1 μm), reported to potentiate BKCa current via the channels β‐subunit, caused a greater effect on whole cell K+ currents in cerebral vessel smooth muscle cells (SMCs) compared to those of cremaster muscle. In contrast, the α‐subunit‐selective BKCa opener, NS‐1619 (20 μm), exerted a similar effect in both preparations. Spontaneously transient outward currents (STOCs) were more apparent (frequency and amplitude) and occurred at more negative membrane potentials in cerebral compared to cremaster SMCs. Also consistent with decreased STOC activity in cremaster SMCs was an absence of detectable Ca2+ sparks (0 of 76 cells) compared to that in cerebral SMCs (76 of 105 cells). Quantitative PCR showed decreased mRNA expression for the β1 subunit and a decrease in the β 1: α ratio in cremaster arterioles compared to cerebral vessels. Similarly, cremaster arterioles showed a decrease in total BKCa protein and the β 1: α‐subunit ratio. The data support vascular heterogeneity with respect to the activity of BKCa in terms of both β‐subunit regulation and interaction with SR‐mediated Ca2+ signalling.

Intracellular mechanisms for alpha 1‐adrenergic regulation of the transient outward current in rabbit atrial myocytes.

1. The intracellular mechanism(s) underlying the decrease of a transient outward K+ current (It) induced by alpha 1‐adrenergic agonists was studied in isolated adult rabbit atrial myocytes using whole‐cell voltage clamp and cell‐attached patch clamp techniques. Experiments were carried out at 22‐23 degrees C. 2. Application of the specific alpha 1‐adrenergic agonist, methoxamine, produced a decrease in It which was irreversible after the non‐hydrolysable GTP analogues, GTP gamma S and Gpp(NH)p, had been introduced into cells via the recording micropipette. 3. Pre‐treatment of cells with 0.1‐0.15 microgram/ml pertussis toxin (PT) for 8‐9 h at 30‐34 degrees C did not prevent the alpha 1‐induced decrease in It. Yet, this protocol, as measured by the PT‐catalysed incorporation of [32P]ADP‐ribose in membrane‐associated 40 and 41 kDa proteins, effectively caused the ADP‐ribosylation of approximately 70% of the PT‐sensitive GTP‐binding proteins (i.e. Gi) in these treated cells. After taking into account the proportion of non‐viable cells (20‐30%), the effectiveness of this treatment probably approaches 100% in the viable myocytes from which electrophysiological recordings were made. 4. Cell‐attached patch recordings showed that bath application of methoxamine altered the single‐channel events underlying It by decreasing their opening probability. Averaged currents from ensemble single‐channel openings recorded in the presence of 0.2 mM‐methoxamine outside the patch reproduced the features of alpha 1‐adrenergic modulation of the macroscopic It observed during whole‐cell voltage clamp measurements. This observation provides evidence for the involvement of a diffusible intracellular second messenger in the alpha 1‐adrenergic modulation of It. 5. The protein kinase C (PKC) activators, 4 beta‐phorbol 12‐myristate 13‐acetate (PMA) and 1‐oleoyl‐2‐acetylglycerol (OAG) increased It, when included in the bath perfusate, whereas the inactive analogues, 4 alpha‐phorbol and 4 alpha‐phorbol 12,13‐didecanoate, had no effect on It. 6. Exposure of cells to the PKC inhibitors, staurosporine and H‐7, either by bath superfusion or intracellularly, via the recording micropipette, did not block the decrease in It produced by methoxamine. 7. Prolonged stimulation of atrial myocytes for 7‐9 h at 22 degrees C with 500 nM‐PMA produced a ‘down‐regulation’ of endogenous PKC activity, as well as a physical loss of the immunoreactive enzyme, as measured by an in vitro assay, and an anti‐PKC monoclonal antibody, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Contribution of potential EF hand motifs to the calcium‐dependent gating of a mouse brain large conductance, calcium‐sensitive K+ channel

1 The large conductance, calcium‐sensitive K+ channel (BKCa channel) is a unique member of the K+‐selective ion channel family in that activation is dependent upon both direct calcium binding and membrane depolarization. Calcium binding acts to dynamically shift voltage‐dependent gating in a negative or left‐ward direction, thereby adjusting channel opening to changes in cellular membrane potential. 2 We hypothesized that the intrinsic calcium‐binding site within the BKCa channel α subunit may contain an EF hand motif, the most common, naturally occurring calcium binding structure. Following identification of six potential sites, we introduced a single amino acid substitution (D/E to N/Q or A) at the equivalent of the ‐z position of a bona fide EF hand that would be predicted to lower calcium binding affinity at each of the six sites. 3 Using macroscopic current recordings of wild‐type and mutant BKCa channels in excised inside‐out membrane patches from HEK 293 cells, we observed that a single point mutation in the C‐terminus (Site 6, FLD923QD to N), adjacent to the ‘calcium bowl’ described by Salkoff and colleagues, shifted calcium‐sensitive gating right‐ward by 50‐65 mV over the range of 2‐12 μM free calcium, but had little effect on voltage‐dependent gating in the absence of calcium. Combining this mutation at Site 6 with a similar mutation at Site 1 (PVD81EK to N) in the N‐terminus produced a greater shift (70‐90 mV) in calcium‐sensitive gating over the same range of calcium. We calculated that these combined mutations decreased the apparent calcium binding affinity ≈11‐fold (129.5 μM vs. 11.3 μM) compared to the wild‐type channel. 4 We further observed that a bacterially expressed protein encompassing Site 6 of the BKCa channel C‐terminus and bovine brain calmodulin were both able to directly bind 45Ca2+ following denaturation and polyacrylamide gel electrophoresis (e.g. SDS‐PAGE). 5 Our results suggest that two regions within the mammalian BKCa channel α subunit, with sequence similarities to an EF hand motif, functionally contribute to the calcium‐sensitive gating of this channel.

Potentiation of large conductance, Ca2+-activated K+ (BK) channels by α5β1 integrin activation in arteriolar smooth muscle

Injury/degradation of the extracellular matrix (ECM) is associated with vascular wall remodelling and impaired reactivity, a process in which altered ECM–integrin interactions play key roles. Previously, we found that peptides containing the RGD integrin‐binding sequence produce sustained vasodilatation of rat skeletal muscle arterioles. Here, we tested the hypothesis that RGD ligands work through α5β1 integrin to modulate the activity of large conductance, Ca2+‐activated K+ (BK) channels in arteriolar smooth muscle. K+ currents were recorded in single arteriolar myocytes using whole‐cell and single‐channel patch clamp methods. Activation of α5β1 integrin by an appropriate, insoluble α5β1 antibody resulted in a 30–50% increase in the amplitude of iberiotoxin (IBTX)‐sensitive, whole‐cell K+ current. Current potentiation occurred 1–8 min after bead–antibody application to the cell surface. Similarly, the endogenous α5β1 integrin ligand fibronectin (FN) potentiated IBTX‐sensitive K+ current by 26%. Current potentiation was blocked by the c‐Src inhibitor PP2 but not by PP3 (0.1–1 μm). In cell‐attached patches, number of open channels × open probability (NPo) of a 230–250 pS K+ channel was significantly increased after FN application locally to the external surface of cell‐attached patches through the recording pipette. In excised, inside‐out patches, the same method of FN application led to large, significant increases in NPo and caused a leftward shift in the NPo–voltage relationship at constant [Ca2+]. PP2 (but not PP3) nearly abolished the effect of FN on channel activity, suggesting that signalling between the integrin and channel involved an increase in Ca2+sensitivity of the channel via a membrane‐delimited pathway. The effects of α5β1 integrin activation on both whole‐cell and single‐channel BK currents could be reproduced in HEK 293 cells expressing the BK channel α‐subunit. This is the first demonstration at the single‐channel level that integrin signalling can regulate an ion channel. Our results show that α5β1 integrin activation potentiates BK channel activity in vascular smooth muscle through both Ca2+‐ and c‐Src‐dependent mechanisms. This mechanism is likely to play a role in the arteriolar dilatation and impaired vascular reactivity associated with ECM degradation.

A Catalytically Inactive Mutant of Type I cGMP-dependent Protein Kinase Prevents Enhancement of Large Conductance, Calcium-sensitive K+ Channels by Sodium Nitroprusside and cGMP

The activation of large conductance, calcium-sensitive K+ (BKCa) channels by the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle. In HEK 293 cells transiently transfected with BKCa channels, we observed that the NO donor sodium nitroprusside and the membrane-permeable analog of cGMP, dibutyryl cGMP, were both able to enhance BKCa channel activity 4–5-fold in cell-attached membrane patches. This enhancement correlated with an endogenous cGMP-dependent protein kinase activity and the presence of the α isoform of type I cGMP-dependent protein kinase (cGKI). We observed that co-transfection of cells with BKCa channels and a catalytically inactive (“dead”) mutant of human cGKIα prevented enhancement of BKCa channel in response to either sodium nitroprusside or dibutyryl cGMP in a dominant negative fashion. In contrast, expression of wild-type cGKIα supported enhancement of channel activity by these two agents. Importantly, both endogenous and expressed forms of cGKIα were found to associate with BKCa channel protein, as demonstrated by a reciprocal co-immunoprecipitation strategy. In vitro, cGKIα was able to directly phosphorylate immunoprecipitated BKCa channels, suggesting that cGKIα-dependent phosphorylation of BKCa channels in situ may be responsible for the observed enhancement of channel activity. In summary, our data demonstrate that cGKIα alone is sufficient to promote the enhancement of BKCa channels in situ after activation of the NO/cGMP signaling pathway.