Rationale Sympathetic stimulation of the heart increases the force of contraction and rate of ventricular relaxation by triggering PKA-dependent phosphorylation of proteins that regulate intracellular calcium. We hypothesized that scaffolding of cAMP signaling complexes by AKAP5 is required for efficient sympathetic stimulation of calcium transients. Objective We examined the function of AKAP5 in the β-adrenergic signaling cascade. Methods and Results We used calcium imaging and electrophysiology to examine the sympathetic response of cardiomyocytes isolated from wild type and AKAP5 mutant animals. The β-adrenergic regulation of calcium transients and the phosphorylation of substrates involved in calcium handling were disrupted in AKAP5 knockout cardiomyocytes. The scaffolding protein, AKAP5 (also called AKAP150/79), targets adenylyl cyclase, PKA, and calcineurin to a caveolin 3-associated complex in ventricular myocytes that also binds a unique sub-population of Cav1.2 L-type calcium channels. Only the caveolin 3-associated Cav1.2 channels are phosphorylated by PKA in response to sympathetic stimulation in wild type heart. However, in the AKAP5 knockout heart the organization of this signaling complex is disrupted, adenylyl cyclase 5/6 no longer associates with caveolin 3 in the T-tubules, and non-caveolin 3 associated calcium channels become phosphorylated after β-adrenergic stimulation although this does not lead to an enhanced calcium transient. The signaling domain created by AKAP5 is also essential for the PKA-dependent phosphorylation of ryanodine receptors and phospholambam. Conclusions These findings identify an AKAP5-organized signaling module that is associated with caveolin 3 and is essential for sympathetic stimulation of the calcium transient in adult heart cells.

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Voltage-gated K؉ (Kv) channels control the excitability of arterial smooth muscle. However, the molecular mechanisms regulating Kv channel function in smooth muscle remain unclear. We examined the hypothesis that the vasoactive peptide angiotensin II (Ang II) regulates arterial smooth muscle Kv channel function via calcineurin-dependent activation of the transcription factor NFAT. We found that sustained administration of Ang II decreased Kv currents (I Kv ) by reducing the expression of Kv2.1 K ؉ channel subunits. This effect of Ang II was independent of pressure but required Ca 2؉ influx through L-type Ca 2؉ channels. Consistent with our hypothesis, we found that calcineurin and NFATc3 are obligatory components of the signaling cascade mediating reduced I Kv by Ang II. We conclude that sustained Ang II exposure increases smooth muscle Ca 2؉ , which leads to activation of calcineurin and NFATc3, culminating in decreased Kv2.1 expression and reduced I Kv function. These results support the novel concept that NFATc3 controls the excitability of arterial smooth muscle by regulating Kv2.1 expression.

Estrogens and xenoestrogens have non-genomic effects mediated by plasma membrane receptors unrelated to the nuclear estrogen receptor (1). One of these is to increase the NP o of BK 1 channels (2), Ca 2ϩ -sensitive members of the voltage-gated K ϩ channel superfamily with important functions in many cells (3). BK channels are composed of ␣ and ␤ subunits. The ␣ subunit forms the K ϩ -selective pore, while ␤ subunits influence the pharmacology, kinetics, and voltage/Ca 2ϩ -sensitivity of BK channels. The ␤1 subunit of smooth muscle BK channels is physiologically important because knockout mice lacking this subunit are hypertensive and demonstrate altered vascular reactivity (4, 5). Recent studies suggest that BK channels are potential targets for 17␤E and xenoestrogens. BK channel NP o is increased by 17␤E, an effect that requires the regulatory ␤1 subunit (2). 17␤E and xenoestrogens reduce coronary vascular tone by inhibiting L-type Ca 2ϩ channels and activating BK channels (6). The pharmacological nature of the putative 17␤E-binding site on the smooth muscle BK ␤1 subunit is unknown. The xenoestrogen Tx, a commonly used chemotherapeutic agent, is an antagonist of the nuclear estrogen receptor (7). It is not known, however, if this clinically important drug increases BK channel NP o . We investigated whether Tx increases BK channel NP o in smooth muscle cells and whether the ␤1 subunit is important for this effect. These findings give insight into BK channel structure and function, non-genomic regulation by xenoestrogens, and Tx-induced side effects. MATERIALS AND METHODSCell Isolation and Preparation-Smooth muscle cells were isolated by enzymatic dispersion described previously (8). Dogs were anesthetized with ketamine, and the colon was removed via a midline incision. Mice were anesthetized with chloroform and killed by cervical dislocation. Human tissue samples were obtained from consenting patients undergoing gastric bypass for the treatment of morbid obesity. Circular muscle of the canine colon and human jejunum was dissected free of mucosa, submucosa, and longitudinal muscle in Ca 2ϩ -free Hanks solution. Strips of muscle were treated with collagenase (345 units/ml; Worthington Biochemical Corp.; Freehold, NJ) in Ca 2ϩ -free Hanks at 37°C to produce suspensions of single cells by gentle stirring. Mouse colon (circular and longitudinal muscle layers) was dissected free of mucosa prior to enzymatic dispersion. Mouse aorta and canine mesenteric vein were enzymatically digested without further dissection.HEK293 cells (ATCC cell line number CRL-1573; Manassas, VA) were grown in glutamax-supplemented RPMI medium (Life Technologies, Inc., Manassas, VA) with 10% heat-inactivated horse serum (Summit Biotechnology; Fort Collins, CO) in a humidified atmosphere with 5% CO 2 at 37°C. cDNA encoding the ␣ and ␤1 subunits from canine colonic smooth was cloned into the pZEOSV mammalian expression vector (Invitrogen; Carlsbad, CA) as described previously (9). Cells were transiently transfected via electroporation with a tot...

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