Channels In Myocytes Research Articles

Regulation of Cardiac Conduction and Arrhythmias by Ankyrin/Spectrin-Based Macromolecular Complexes.

The cardiac conduction system is an extended network of excitable tissue tasked with generation and propagation of electrical impulses to signal coordinated contraction of the heart. The fidelity of this system depends on the proper spatio-temporal regulation of ion channels in myocytes throughout the conduction system. Importantly, inherited or acquired defects in a wide class of ion channels has been linked to dysfunction at various stages of the conduction system resulting in life-threatening cardiac arrhythmia. There is growing appreciation of the role that adapter and cytoskeletal proteins play in organizing ion channel macromolecular complexes critical for proper function of the cardiac conduction system. In particular, members of the ankyrin and spectrin families have emerged as important nodes for normal expression and regulation of ion channels in myocytes throughout the conduction system. Human variants impacting ankyrin/spectrin function give rise to a broad constellation of cardiac arrhythmias. Furthermore, chronic neurohumoral and biomechanical stress promotes ankyrin/spectrin loss of function that likely contributes to conduction disturbances in the setting of acquired cardiac disease. Collectively, this review seeks to bring attention to the significance of these cytoskeletal players and emphasize the potential therapeutic role they represent in a myriad of cardiac disease states.

Large conductance voltage- and calcium-gated potassium channels (BK) in cerebral artery myocytes of perinatal fetal primates share several major characteristics with the adult phenotype.

Large conductance voltage- and calcium-gated channels (BK) control fundamental processes, including smooth muscle contractility and artery diameter. We used a baboon (Papio spp) model of pregnancy that is similar to that of humans to characterize BK channels in the middle cerebral artery and its branches in near-term (165 dGa) primate fetuses and corresponding pregnant mothers. In cell-attached patches (K+pipette = 135 mM) on freshly isolated fetal cerebral artery myocytes, BK currents were identified by large conductance, and voltage- and paxilline-sensitive effects. Their calcium sensitivity was confirmed by a lower Vhalf (transmembrane voltage needed to reach half-maximal current) in inside-out patches at 30 versus 3 μM [Ca2+]free. Immunostaining against the BK channel-forming alpha subunit revealed qualitatively similar levels of BK alpha protein-corresponding fluorescence in fetal and maternal myocytes. Fetal and maternal BK currents recorded at 3 μM [Ca2+]free from excised membrane patches had similar unitary current amplitude, and Vhalf. However, subtle differences between fetal and maternal BK channel phenotypes were detected in macroscopic current activation kinetics. To assess BK function at the organ level, fetal and maternal artery branches were pressurized in vitro at 30 mmHg and probed with the selective BK channel blocker paxilline (1 μM). The degree of paxilline-induced constriction was similar in fetal and maternal arteries, yet the constriction of maternal arteries was achieved sooner. In conclusion, we present a first identification and characterization of fetal cerebral artery BK channels in myocytes from primates. Although differences in BK channels between fetal and maternal arteries exist, the similarities reported herein advance the idea that vascular myocyte BK channels are functional near-term, and thus may serve as pharmacological targets during the perinatal-neonatal period.

Interventricular differences in sodium current and its potential role in Brugada syndrome.

Brugada syndrome (BrS) is an inherited disease associated with ST elevation in the right precordial leads, polymorphic ventricular tachycardia (PVT), and sudden cardiac death in adults. Mutations in the cardiac sodium channel account for a large fraction of BrS cases. BrS manifests in the right ventricle (RV), which led us to examine the biophysical and molecular properties of sodium channel in myocytes isolated from the left (LV) and right ventricle. Patch clamp was used to record sodium current (I Na) in single canine RV and LV epicardial (epi) and endocardial (endo) myocytes. Action potentials were recorded from multicellular preparations and single cells. mRNA and proteins were determined using quantitative RT‐PCR and Western blot. Although LV wedge preparations were thicker than RV wedges, transmural ECG recordings showed no difference in the width of the QRS complex or transmural conduction time. Action potential characteristics showed RV epi and endo had a lower V max compared with LV epi and endo cells. Peak I Na density was significantly lower in epi and endo RV cells compared with epi and endo LV cells. Recovery from inactivation of I Na in RV cells was slightly faster and half maximal steady‐state inactivation was more positive. β2 and β4 mRNA was detected at very low levels in both ventricles, which was confirmed at the protein level. Our observations demonstrate that V max and Na+ current are smaller in RV, presumably due to differential Nav1.5/β subunit expression. These results provide a potential mechanism for the right ventricular manifestation of BrS.

Arterial smooth muscle cell PKD2 (TRPP1) channels control systemic blood pressure

Hypertension, a disease that afflicts more than one quarter of the World's population, is a major risk factor for cardiovascular diseases. Blood pressure is determined in part by arterial smooth muscle cells (myocytes) that alter resistance vessel tone. In vivo mechanisms that regulate myocyte contractility to control physiological blood pressure and become pathological during hypertension are poorly understood. Several Transient Receptor Potential (TRP) channels are proposed to be expressed in arterial myocytes, although it is unclear if these proteins control physiological blood pressure and can be targeted to alleviate hypertension. Here, we generated the first inducible, smooth muscle‐specific knockout for a TRP channel, namely for PKD2 (TRPP1), to investigate blood pressure regulation by this protein. Data indicate that PKD2 knockout dilates resistance‐size systemic arteries and reduces blood pressure. We show that heterogeneous stimuli activate PKD2 channels in arteries of different organs. Intravascular pressure stimulate PKD2 channels in skeletal muscle arterial myocytes, whereas α1‐adrenergic receptors activate PKD2 channels in myocytes of mesenteric arteries. Regardless of the stimulus or arterial bed, PKD2 current activation in myocytes leads to vasoconstriction. Hypertension is associated with an increase in the abundance of plasma membrane PKD2 channels in systemic arteries. Myocyte‐specific PKD2 knockout caused vasodilation, lowered systemic blood pressure and prevented arterial remodeling during hypertension. In summary, we show that PKD2 channels are activated by distinct vasoconstrictor stimuli in arterial myocytes of different tissues, control physiological systemic blood pressure, are upregulated during hypertension and knockout reduces high blood pressure. These data suggest that targeting of arterial myocyte PKD2 channels could be exploited to control blood pressure and alleviate cardiovascular diseases.Support or Funding InformationThis study was supported by NIH/NHLBI grants HL67061, 133256 and HL137745 to J.H.J, and American Heart Association Scientist Development Grants to S.B and M.D.L.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effects of Severe Iron Deficiency Anemia on T-wave Intervals

Aim: Changes in the iron level in the myocardium may be affected by sodium and potassium channels in myocytes and there may be a contribution to the etiology of cardiac arrhythmia. The aim of this study was to examine for the first time in literature how the Tp-e interval, which has been accepted in recent years as a strong ventricular repolarisation index, is affected in female patients with severe iron deficiency anemia. Material and Methods: The study included a total of 64 participants, as a study group of 34 patients diagnosed with severe iron deficiency anemia (Hgb<8g/dL Ferritin<14 mcg/L, MCV<80 mm3) and a control group of 34 subjects. To determine the QTc and Tp-Te interval durations, manual examination was made of manual precordial V5 lead ECGs taken routinely on the presentation of all participants. Results: The Tp-e interval was determined to be statistically significantly shorter in the patients (78.97 ± 8.63 ms) compared to the control group (84.57 ± 10.13 ms) (p<0.05). A positive relationship was determined between Tp-e and QTc durations and Hgb and ferritin levels (p<0.05). Conclusion: The results of the study showed that the QTc duration and the Tp-e duration were shorter in female patients with iron deficiency anemia, and therefore iron deficiency anemia can be discounted as a cause of prolonged QTc and Tp-e durations.

Aerobic exercise of low to moderate intensity corrects unequal changes in BK(Ca) subunit expression in the mesenteric arteries of spontaneously hypertensive rats.

Accumulating evidence indicates that hypertension is associated with "ion channel remodeling" of vascular smooth muscle cells (VSMCs). The objective of this study was to determine the effects of exercise intensity/volume on hypertension-associated changes in large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in mesenteric arteries (MAs) from spontaneously hypertensive rats (SHR). Male SHRs were randomly assigned to three groups: a low-intensity aerobic exercise group (SHR-L: 14 m/min), a moderate-intensity aerobic exercise group (SHR-M: 20 m/min), and a sedentary group (SHR). Age-matched Wistar-Kyoto rats (WKYs) were used as normotensive controls. Exercise groups completed an 8-week exercise program. Elevation of the alpha and beta1 proteins was unequal in MA myocytes from SHRs, with the beta1 subunit increasing more than the alpha subunit. BK(Ca) contribution to vascular tone regulation was higher in the myocytes and arteries of SHRs compared to WKYs. SHR BK(Ca) channel subunit protein expression, beta1/alpha ratio, whole cell current density and single-channel open probability was also increased compared with WKYs. Aerobic exercise lowered systemic blood pressure and normalized hypertension-associated BK(Ca) alterations to normotensive control levels in the SHRs. These effects were more pronounced in the moderate-intensity group than in the low-intensity group. There is a dose-effect for aerobic exercise training in the range of low to moderate-intensity and accompanying volume for the correction of the pathological adaptation of BK(Ca) channels in myocytes of MAs from SHR.

A Role of BK Channel in Regulation of Ca2+ Channel in Ventricular Myocytes by Substrate Stiffness

Substrate stiffness is crucial for diverse cell functions, but the mechanisms conferring cells with mechanosensitivity are still elusive. By tailoring substrate stiffness with 10-fold difference, we showed that L-type voltage-gated Ca2+ channel current density was greater in chick ventricular myocytes cultured on the stiff substrate than on the soft substrate. Blockage of the BK channel increased the Ca2+ current density on the soft substrate and consequently eliminated substrate stiffness regulation of the Ca2+ channel. The expression of the BK channel, including the STREX-containing α-subunit that forms stretch-activated BK channel in myocytes and the BK channel function in myocytes (and also in HEK293 cells heterologously expressing STREX-containing α- and β1-subunits) was reduced in cells cultured on the stiff substrate. Furthermore, in HEK293 cells coexpressing the cardiac CaV1.2 channel and STREX-containing BK channel, the Ca2+ current density was greater in cells on the stiff substrate, which was not observed in cells expressing the CaV1.2 channel alone or coexpressing with the STREX-deleted BK channel. These results provide strong evidence to show that the stretch-activated BK channel plays a key role in functional regulation of cardiac voltage-gated Ca2+ channel by substrate stiffness, revealing, to our knowledge, a novel mechanosensing mechanism in ventricular myocytes.

Angiotensin II reduces the surface abundance of KV 1.5 channels in arterial myocytes to stimulate vasoconstriction.

Several different voltage-dependent K+ (KV ) channel isoforms are expressed in arterial smooth muscle cells (myocytes). Vasoconstrictors inhibit KV currents, but the isoform selectivity and mechanisms involved are unclear. We show that angiotensin II (Ang II), a vasoconstrictor, stimulates degradation of KV 1.5, but not KV 2.1, channels through a protein kinase C- and lysosome-dependent mechanism, reducing abundance at the surface of mesenteric artery myocytes. The AngII-induced decrease in cell surface KV 1.5 channels reduces whole-cell KV 1.5 currents and attenuates KV 1.5 function in pressurized arteries. We describe a mechanism by which AngII stimulates protein kinase C-dependent KV 1.5 channel degradation, reducing the abundance of functional channels at the myocyte surface. Smooth muscle cells (myocytes) of resistance-size arteries express several different voltage-dependent K+ (KV ) channels, including KV 1.5 and KV 2.1, which regulate contractility. Myocyte KV currents are inhibited by vasoconstrictors, including angiotensinII (AngII), but the mechanisms involved are unclear. Here, we tested the hypothesis that AngII inhibits KV currents by reducing the plasma membrane abundance of KV channels in myocytes. AngiotensinII (applied for 2h) reduced surface and total KV 1.5 protein in rat mesenteric arteries. In contrast, AngII did not alter total or surface KV 2.1, or KV 1.5 or KV 2.1 cellular distribution, measured as the percentage of total protein at the surface. Bisindolylmaleimide (BIM; a protein kinaseC blocker), a protein kinaseC inhibitory peptide or bafilomycinA (a lysosomal degradation inhibitor) each blocked the AngII-induced decrease in total and surface KV 1.5. Immunofluorescence also suggested that AngII reduced surface KV 1.5 protein in isolated myocytes; an effect inhibited by BIM. Arteries were exposed to AngII or AngII plus BIM (for 2h), after which these agents were removed and contractility measurements performed or myocytes isolated for patch-clamp electrophysiology. AngiotensinII reduced both whole-cell KV currents and currents inhibited by Psora-4, a KV 1.5 channel blocker. AngiotensinII also reduced vasoconstriction stimulated by Psora-4 or 4-aminopyridine, another KV channel inhibitor. These data indicate that AngII activates protein kinaseC, which stimulates KV 1.5 channel degradation, leading to a decrease in surface KV 1.5, a reduction in whole-cell KV 1.5 currents and a loss of functional KV 1.5 channels in myocytes of pressurized arteries.

Local coupling of TRPC6 to ANO1/TMEM16A channels in smooth muscle cells amplifies vasoconstriction in cerebral arteries.

Anoctamin-1 [ANO1, also known as transmembrane protein 16A (TMEM16A)] is a Ca(2+)-activated Cl(-) channel expressed in arterial myocytes that regulates membrane potential and contractility. Signaling mechanisms that control ANO1 activity in arterial myocytes are poorly understood. In cerebral artery myocytes, ANO1 channels are activated by local Ca(2+) signals generated by plasma membrane nonselective cation channels, but the molecular identity of these proteins is unclear. Arterial myocytes express several different nonselective cation channels, including multiple members of the transient receptor potential receptor (TRP) family. The goal of this study was to identify localized ion channels that control ANO1 currents in cerebral artery myocytes. Coimmunoprecipitation and immunofluorescence resonance energy transfer microscopy experiments indicate that ANO1 and canonical TRP 6 (TRPC6) channels are present in the same macromolecular complex and localize in close spatial proximity in the myocyte plasma membrane. In contrast, ANO1 is not near TRPC3, TRP melastatin 4, or inositol trisphosphate receptor 1 channels. Hyp9, a selective TRPC6 channel activator, stimulated Cl(-) currents in myocytes that were blocked by T16Ainh-A01, an ANO1 inhibitor, ANO1 knockdown using siRNA, and equimolar replacement of intracellular EGTA with BAPTA, a fast Ca(2+) chelator that abolishes local Ca(2+) signaling. Hyp9 constricted pressurized cerebral arteries, and this response was attenuated by T16Ainh-A01. In contrast, T16Ainh-A01 did not alter depolarization-induced (60 mM K(+)) vasoconstriction. These data indicate that TRPC6 channels generate a local intracellular Ca(2+) signal that activates nearby ANO1 channels in myocytes to stimulate vasoconstriction.

Rab25 influences functional Cav1.2 channel surface expression in arterial smooth muscle cells

Plasma membrane-localized CaV1.2 channels are the primary calcium (Ca(2+)) influx pathway in arterial smooth muscle cells (myocytes). CaV1.2 channels regulate several cellular functions, including contractility and gene expression, but the trafficking pathways that control the surface expression of these proteins are unclear. Similarly, expression and physiological functions of small Rab GTPases, proteins that control vesicular trafficking in arterial myocytes, are poorly understood. Here, we investigated Rab proteins that control functional surface abundance of CaV1.2 channels in cerebral artery myocytes. Western blotting indicated that Rab25, a GTPase previously associated with apical recycling endosomes, is expressed in cerebral artery myocytes. Immunofluorescence Förster resonance energy transfer (immunoFRET) microscopy demonstrated that Rab25 locates in close spatial proximity to CaV1.2 channels in myocytes. Rab25 knockdown using siRNA reduced CaV1.2 surface and intracellular abundance in arteries, as determined using arterial biotinylation. In contrast, CaV1.2 was not located nearby Rab11A or Rab4 and CaV1.2 protein was unaltered by Rab11A or Rab4A knockdown. Rab25 knockdown resulted in CaV1.2 degradation by a mechanism involving both lysosomal and proteasomal pathways and reduced whole cell CaV1.2 current density but did not alter voltage dependence of current activation or inactivation in isolated myocytes. Rab25 knockdown also inhibited depolarization (20-60 mM K(+)) and pressure-induced vasoconstriction (myogenic tone) in cerebral arteries. These data indicate that Rab25 is expressed in arterial myocytes where it promotes surface expression of CaV1.2 channels to control pressure- and depolarization-induced vasoconstriction.

Developmental acceleration of bradykinin-dependent relaxation by prenatal chronic hypoxia impedes normal development after birth.

Bradykinin-induced activation of the pulmonary endothelium triggers nitric oxide production and other signals that cause vasorelaxation, including stimulation of large-conductance Ca(2+)-activated K(+) (BKCa) channels in myocytes that hyperpolarize the plasma membrane and decrease intracellular Ca(2+). Intrauterine chronic hypoxia (CH) may reduce vasorelaxation in the fetal-to-newborn transition and contribute to pulmonary hypertension of the newborn. Thus we examined the effects of maturation and CH on the role of BKCa channels during bradykinin-induced vasorelaxation by examining endothelial Ca(2+) signals, wire myography, and Western immunoblots on pulmonary arteries isolated from near-term fetal (∼ 140 days gestation) and newborn, 10- to 20-day-old, sheep that lived in normoxia at 700 m or in CH at high altitude (3,801 m) for >100 days. CH enhanced bradykinin-induced relaxation of fetal vessels but decreased relaxation in newborns. Endothelial Ca(2+) responses decreased with maturation but increased with CH. Bradykinin-dependent relaxation was sensitive to 100 μM nitro-L-arginine methyl ester or 10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, supporting roles for endothelial nitric oxide synthase and soluble guanylate cyclase activation. Indomethacin blocked relaxation in CH vessels, suggesting upregulation of PLA2 pathways. BKCa channel inhibition with 1 mM tetraethylammonium reduced bradykinin-induced vasorelaxation in the normoxic newborn and fetal CH vessels. Maturation reduced whole cell BKCa channel α1-subunit expression but increased β1-subunit expression. These results suggest that CH amplifies the contribution of BKCa channels to bradykinin-induced vasorelaxation in fetal sheep but stunts further development of this vasodilatory pathway in newborns. This involves complex changes in multiple components of the bradykinin-signaling axes.

Identification of Voltage‐Activated Calcium Currents in Renal Afferent and Efferent Arterioles of the Rat

Based on indirect methods, it is suggested that both L‐ and T‐type Ca channels mediate signaling in the renal afferent arteriole (AA) and that T‐type Ca channels are involved in signaling in the efferent arteriole (EA). Our study was initiated to characterize Ca currents in these vessels, directly, using patch clamp. Native myocytes were isolated from individually isolated rat AA and EA and from tail arteries. Inward currents were measured in physiologic 1.5 mmol/L Ca and 10 mmol/L Ba using the whole‐cell configuration. By exploiting known differences in activation and inactivation properties and sensitivities to nifedipine and kurtoxin, we could readily demonstrate the presence of both L‐ and T‐type Ca channels in myocytes from the rat tail artery. AA myocytes exhibited relatively large Ca current densities (−2.0±0.2 pA/pF), which increased 3.6 fold in Ba. These currents were blocked by nifedipine, but not by kurtoxin or mibefradil and did not exhibit the activation and inactivation characteristics of T‐type Ca channels, compared to tail artery myocytes. EA myocytes did not exhibit a marked voltage‐activated inward current in 1.5 mmol/L Ca. Thus, our findings support the physiologic role of L‐type Ca channels in the AA, but not EA, and do not support the premise that T‐type Ca channels are significantly present in either vessel. Supported by the Royal Society and the Alberta Heritage Foundation for Medical Research.

Beta Subunit Transmembrane Domains Determine the Differential Ethanol Responses of β1- vs. β4-Containing BK Channels

Ethanol (10-100 mM) usually decreases the steady-state activity (Po) of vascular smooth muscle BK channels while increasing Po of neuronal BK channels (Brodie et al., 2007). Native BK complexes consist of channel-forming α and tissue-specific, accessory β subunits. Four β subunit types were identified, with β1 and β4 prevailing in smooth muscle and neurons, respectively (Brenner et al., 2000). Remarkably, differential expression of β1- vs. β4-containing BK channels modifies Po adaptation to protracted ethanol exposure (Feinberg-Zadek et al., 2008; Martin et al., 2008). On the other hand, β1 is necessary for ethanol to inhibit vascular smooth muscle BK channels and the resulting vasoconstriction (Bukiya et al., 2009). To begin to address the mechanisms and protein regions contributing to differential ethanol actions on BK channels, we expressed channel-forming cbv1 subunits in absence and presence of β1 or β4. Then, we conducted G/Gmax-V plots from ionic currents in inside-out patches and evaluated their modification by acute ethanol exposure. As described for other BK channels (Orio et al., 2002), β1 but not β4 significantly reduced cbv1 V0.5 across 1-1,000 μM calcium. Ethanol (50 mM) decreased homomeric cbv1 V0.5 at low calcium while increasing V0.5 at high calcium, the “crossover” from current potentiation to inhibition occurring at 20 μM calcium. This crossover was left-shifted to 2 μM by β1 co-expression but unaltered by β4 co-expression. Therefore, β1 enables ethanol inhibition of current at calcium levels that are reached near the BK channel in myocytes. Evaluation of G/Gmax-V plots from currents mediated by cbv1±chimeric β1/β4 demonstrated that ethanol responses of complexes containing β chimeras that included β1 TM domains mimicked cbv1+wtβ1 responses. Conversely, ethanol responses of complexes containing β chimeras that included β4 TM domains mimicked cbv1+wtβ4 responses. Support:R37-AA011560 (AMD).

Identification of Sterol-Sensing Amino Acids in Transmembrane Domain 2 of the BK Channel Beta 1 subunit

The activity of large conductance, voltage- and calcium-gated potassium (BK) channels in myocytes critically controls arterial diameter. At <300 μM in the aqueous phase (below critical micellar concentration), lithocholate (LC) and related steroids increase myocyte BK channel activity (Po) and cause arterial dilation. We showed that: 1) BK β1 TM2 is critical for the channel sensitivity to LC, and 2) BK β2-4 cannot substitute for β1 in providing LC-sensitivity to BK channels (Bukiya et al., 2008; 2009). Moreover, computational dynamics identified two LC-sensing amino acid clusters in BK β1TM2: L157,L158,T165 and T169,L172,L173. Here, we used pinpoint mutagenesis and patch-clamp electrophysiology to determine the relative contribution of each cluster to the LC-sensitivity of BK channels. BK α (cbv1) and β1 subunits cloned from rat cerebral artery myocytes (AY330293; FJ154955) were coexpressed in Xenopus oocytes, and BK currents were recorded from inside-out patches at Ca2+i=10 μM and Vm=-20 to −40mV. BK β1T165A and β1T165A,L157A,L158A mutants rendered LC-sensitive BK channels, with 150 μM LC-induced increase in Po (×1.7 times) being identical to that found with cbv1+wtβ1. In contrast, BK β1T165A,T169A and β1T169A mutants were absolutely unresponsive to ≥150 μM LC. Data unveil a critical role for β1T169 in providing LC-sensitivity to BK channels. While the β1T169S mutation was able to rescue the channel's LC-sensitivity, the β1T169S,L172A,L173A mutation failed to do so. Moreover, BK β1L172A,L173A was also LC-insensitive. These data validate our computational prediction that hydrogen bonding between T/S169 and the steroid hydroxyl, and hydrophobic interactions between L172,L173 and the steroid rings are both necessary for successful docking of LC onto the T169,L172,L173 cluster. Thus, T169, L172 and L173 in BK β1TM2 provide a docking surface that is essential for the LC-sensitivity of BK channels.Support: HL104631 (AMD).

Autocrine signaling via A1 adenosine receptors causes downregulation of M2 receptors in adult rat atrial myocytes in vitro

G protein-activated K(+) channels composed of Kir3 (GIRK) subunits contribute to regulation of heart rate and excitability. Opening of these channels in myocytes is increased by binding of G(βγ) upon activation of muscarinic M(2) receptors (M(2)-R) or A(1) adenosine receptors (A(1)-R). It has been shown that saturating activation of A(1)-R resulted in a smaller GIRK current than activation of M(2)-R. Adenovirus-driven overexpression of the A(1)-R caused an increase in current induced by adenosine (I(K(Ado))), whereas the M(2)-R-activated current (I(K(ACh))) was reduced. Here, we sought to get deeper insight into the mechanism causing this negative crosstalk. GIRK current in cultured rat atrial myocytes was recorded in whole cell mode. Adenovirus-driven RNA interference targeting the M(2)-R resulted in a reduction in I(K(ACh)) without affecting I(K(Ado)), arguing against a competition of the two receptors for common signaling complexes. The negative effect of A(1)-R overexpression on I(K(ACh)) was reduced by the A(1)-R antagonist DPCPX and augmented by the agonist chloro-N6-cyclopentyladenosin (CCPA). In native myocytes incubation with either CCPA or the muscarinic agonist carbachol resulted in reduction in I(K(ACh)) and I(K(Ado)), suggesting common pathways of A(1)-R and M(2)-R downregulation. In the absence of agonist, inhibition of adenosine deaminase by EHNA or exposure to AMP, less to ADP, but not ATP resulted in reduction of I(K(ACh)) and I(K(Ado)). Our data indicate that atrial myocytes generate adenosine from extracellular AMP, which activates A(1)-R in an autocrine fashion. Chronic activation of A(1)-R causes parallel downregulation of both A(1)-R and M(2)-R.

Isoform-Selective Physical Coupling of TRPC3 Channels to IP 3 Receptors in Smooth Muscle Cells Regulates Arterial Contractility

Inositol 1,4,5-trisphosphate (IP(3))-induced vasoconstriction can occur independently of intracellular Ca(2+) release and via IP(3) receptor (IP(3)R) and canonical transient receptor potential (TRPC) channel activation, but functional signaling mechanisms mediating this effect are unclear. Study mechanisms by which IP(3)Rs stimulate TRPC channels in myocytes of resistance-size cerebral arteries. Immunofluorescence resonance energy transfer (immuno-FRET) microscopy using isoform-selective antibodies indicated that endogenous type 1 IP(3)Rs (IP(3)R1) are in close spatial proximity to TRPC3, but distant from TRPC6 or TRPM4 channels in arterial myocytes. Endothelin-1 (ET-1), a phospholipase C-coupled receptor agonist, elevated immuno-FRET between IP(3)R1 and TRPC3, but not between IP(3)R1 and TRPC6 or TRPM4. TRPC3, but not TRPC6, coimmunoprecipitated with IP(3)R1. TRPC3 and TRPC6 antibodies selectively inhibited recombinant channels, but only the TRPC3 antibody blocked IP(3)-induced nonselective cation current (I(Cat)) in myocytes. TRPC3 knockdown attenuated immuno-FRET between IP(3)R1 and TRPC3, IP(3)-induced I(Cat) activation, and ET-1 and IP(3)-induced vasoconstriction, whereas TRPC6 channel knockdown had no effect. ET-1 did not alter total or plasma membrane-localized TRPC3, as determined using surface biotinylation. RT-PCR demonstrated that C-terminal calmodulin and IP(3)R binding (CIRB) domains are present in myocyte TRPC3 and TRPC6 channels. A peptide corresponding to the IP(3)R N-terminal region that can interact with TRPC channels activated I(Cat). A TRPC3 CIRB domain peptide attenuated IP(3)- and ET-1-induced I(Cat) activation and vasoconstriction. IP(3) stimulates direct coupling between IP(3)R1 and membrane-resident TRPC3 channels in arterial myocytes, leading to I(Cat) activation and vasoconstriction. Close spatial proximity between IP(3)R1 and TRPC3 establishes this isoform-selective functional interaction.

Channel β2–4 subunits fail to substitute for β1 in sensitizing BK channels to lithocholate

Large conductance, calcium- and voltage-gated potassium (BK) channels regulate numerous physiological processes. While most basic functional characteristics of native BK channels are reproduced by BK α ( slo1) subunit homotetramers, key biophysical and pharmacological properties are drastically modified by the presence of auxiliary β subunits (encoded by KCNMB1–4). Numerous physiological steroids, including sex hormones, gluco- and mineralocorticoids, activate β subunit-containing BK channels, yet these steroids appear to be sensed by different types of β subunits, with some steroids being sensed by homomeric slo1 channels as well. We recently showed that β1 sensitizes the BK channel to μM concentrations of lithocholate (LC). Following expression of rat cerebral artery myocyte slo1 subunits (“cbv1”) with β1, β2, β3 or β4 in Xenopus laevis oocytes we now demonstrate that BK β2, β3 and β4 subunits fail to substitute for β1 in providing LC-sensitivity (150 μM) to the BK channel. These findings document for the first time a rather selective steroid activation of BK channels via a particular channel accessory subunit. In addition, LC routinely activated native BK channels in myocytes freshly isolated from rat cerebral artery smooth muscle, where BK β1 is highly expressed, while failing to do so in skeletal ( flexor digitorum brevis) muscle, where BK β1 expression is negligible. This indicates that the native environment of the BK channel sustains the LC-sensitivity distinctly provided to the BK channel by β1 subunits. Our study indicates that LC represents a unique tool to probe the presence of functional β1-subunits and selectively activate BK channels in tissues that highly express KCNMB1.

Endothelin‐1 constricts cerebral arteries by stimulating physical coupling between type 1 IP3 receptors and TRPC3 channels in myocytes

Vasoconstrictors elevate inositol 1,4,5‐trisphophate (IP3), but signaling mechanisms activated by this second messenger in arterial myocytes are poorly understood. Recently, we described that IP3‐induced vasoconstriction can occur independently of intracellular Ca2+ release and via IP3 receptor (IP3R) and TRPC3 channel activation (Xi et al., Circ Res. 2008; 102:1118‐26). Here, we tested the hypothesis that IP3Rs stimulate TRPC channels due to physical coupling between these proteins in cerebral artery myocytes. An IP3R1 antibody co‐immunoprecipitated both IP3R1 and TRPC3 channels from cerebral artery lysate. IP3R1 and TRPC3‐bound fluorescent (Cy2, Cy3) secondary antibodies generated significant immunofluorescence resonance energy transfer (N‐FRET) in myocytes. In contrast, antibodies bound to IP3R1 and TRPM4 generated weak N‐FRET. Endothelin‐1 (ET‐1), a PLC‐coupled receptor agonist, increased N‐FRET between IP3R1 and TRPC3‐bound fluorescent antibodies. A peptide corresponding to the TRPC channel C‐terminus calmodulin and IP3R binding domain (CIRB) reduced ET‐1 and IP3‐induced ICat activation in myocytes. Similarly, a membrane‐permeant (HIV‐1 TAT) CIRB peptide reduced ET‐1‐induced constriction in pressurized arteries. Data suggest that ET‐1 enhances physical coupling between the CIRB domain of IP3R1 and TRPC3, leading to cation current activation and vasoconstriction. R01 HL67061.

The Cardiac IKs Potassium Channel Macromolecular Complex Includes the Phosphodiesterase PDE4D3

The cardiac I(Ks) potassium channel is a macromolecular complex consisting of alpha-(KCNQ1) and beta-subunits (KCNE1) and the A kinase-anchoring protein (AKAP) Yotiao (AKAP-9), which recruits protein kinase A) and protein phosphatase 1 to the channel. Here, we have tested the hypothesis that specific cAMP phosphodiesterase (PDE) isoforms of the PDE4D family that are expressed in the heart are also part of the I(Ks) signaling complex and contribute to its regulation by cAMP. PDE4D isoforms co-immunoprecipitated with I(Ks) channels in hearts of mice expressing the I(Ks) channel. In myocytes isolated from these mice, I(Ks) was increased by pharmacological PDE inhibition. PDE4D3, but not PDE4D5, co-immunoprecipitated with the I(Ks) channel only in Chinese hamster ovary cells co-expressing AKAP-9, and PDE4D3, but not PDE4D5, co-immunoprecipitated with AKAP-9. Functional experiments in Chinese hamster ovary cells expressing AKAP-9 and either PDE4D3 or PDE4D5 isoforms revealed modulation of the I(Ks) response to cAMP by PDE4D3 but not PDE4D5. We conclude that PDE4D3, like protein kinase A and protein phosphatase 1, is recruited to the I(Ks) channel via AKAP-9 and contributes to its critical regulation by cAMP.

Monoaminergic modulation of stretch-activated ion channels in myocytes isolated from the insect visceral muscle

Monoaminergic modulation of stretch-activated ion channels in myocytes isolated from the insect visceral muscle