Original articleLocalization of f-channels to caveolae mediates specific β2-adrenergic receptor modulation of rate in sinoatrial myocytes
Introduction
In the mammalian heart, spontaneous activity is an intrinsic property of pacemaker cells of the SAN. Although generation of rhythmic activity does not require nervous inputs, autonomic innervation of the SAN region is responsible for the modulation of heart rate.
Fine tuning of cardiac rhythm is achieved by the opposing actions of the neurotransmitters norepinephrine and acetylcholine released by sympathetic and parasympathetic branches, respectively, of the autonomic nervous system. Activated adrenergic and muscarinic receptors, acting through different G-proteins, stimulate and inhibit adenylate cyclase causing an increase and a decrease in cytoplasmic cAMP concentration, respectively.
To date, three different subtypes of β-ARs are known (i.e., β1, β2, and β3). Only β1- and β2-ARs are abundantly expressed in cardiac tissue, with a predominance of the β1-ARs subtype. In mammals, the β1/β2 ratio varies among different cardiac regions: it is very high in the ventricles, lower in the atria and lower still in the SAN. The distribution pattern varies somewhat in different species but the trend is the same, with the SAN being the region with the highest level of expression of β2-ARs [1], [2], [3], [4].
In the heart, functional coupling to adenylate cyclase is reported to be more efficient for β2- than for β1-adrenergic receptors [4], [5], [6]. Although this difference could be due simply to the intrinsic properties of receptors, it could also result from differences in the subcellular distribution of receptors and the specific signal transduction proteins to which each type of receptor is coupled.
There are indeed indications that different β-adrenergic receptors have different subcellular localizations. In rat ventricular myocytes, for example, β2-ARs are confined to caveolar spaces along with isoforms V/VI of adenylate cyclase, while β1-ARs are for the most part excluded from the same membrane microdomains [7], [8], [9], [10].
We have recently shown that pacemaker f-channels localize to membrane lipid rafts both in rabbit SAN myocytes and in HEK293 cells expressing HCN4, the major HCN channel isoform contributing to native f-channels [11]. The If current plays a crucial role in the generation and modulation of the slow diastolic depolarization and heart rate [12], [13], [14]. f-channels mediate autonomic modulation of rate by their sensitivity to cAMP, which binds directly to channels and increases the probability of channel opening via a depolarizing shift of the open probability curve [15]; the consequent current increase steepens the diastolic depolarization rate of action potentials and is thus responsible for rate acceleration [12]. We have recently shown that a form of familial sinus bradycardia is associated with a mutation of the HCN4 gene. The mutation causes the channels to activate at more negative voltages than wild-type channels; this effect, similar to the physiologic effect of vagal stimulation, is able to slow heart rate by decreasing the pacemaker current during diastolic depolarization [16].
We hypothesized that, as well as in ventricular myocytes, β2-ARs might be preferentially located in caveolae also in rabbit SAN myocytes, and might therefore colocalize with f-channels in pacemaker cells. If this were the case, adrenergic modulation of If current and of cardiac chronotropism would be expected to depend mostly on stimulation of β2-ARs. We therefore investigated the subcellular distribution of β-ARs and the efficiency of f-channel and rate modulation by β1-AR vs. β2-AR stimulation in rabbit SAN myocytes.
Section snippets
Isolation of rabbit SAN myocyctes
The procedures employed in this work conformed to guidelines for the care and use of laboratory animals as established by State (D.L. 116/1992) and European directives (86/609/CEE). Rabbit SAN myocytes were isolated as previously reported [17], stored at 4 °C in Tyrode solution (in mM: NaCl, 140; KCl, 5.4; CaCl2, 1.8; MgCl2, 1; d-glucose, 5.5; HEPES–NaOH, 5; pH 7.4) and used for the day.
Cholesterol depletion
Membrane cholesterol depletion in SAN myocytes was achieved by 2% methyl-β-cyclodextrin treatment (MβCD,
Results
Previous evidence has indicated that pacemaker f-channels of SAN cells localize to lipid raft-enriched fractions of the plasma membrane [11]. The available data however cannot distinguish between caveolar and non-caveolar lipid rafts. In order to discriminate caveolar from non-caveolar localization of f-channels, we immunoprecipitated proteins from a SAN extract (see Materials and methods) using either anti-caveolin 3 or anti-HCN4 primary antibodies and checked in the precipitated proteins for
Discussion
The role played by funny channels in the generation and autonomic modulation of heart rate is well established [12]. f-channels are directly activated by cAMP [15], whose free cytoplasmic concentration is set at any time by the opposing actions of adenylate cyclase and cAMP phosphodiesterase. Since the activity of adenylate cyclase, and hence the cAMP concentration, are increased by β-adrenergic and decreased by muscarinic stimulation, the cAMP/f-channel-dependent mechanism is well suited to
Acknowledgments
This work was supported by grants from the Ministero dell'Istruzione, Università e Ricerca (FIRB 2003) to DD.
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