The role of ion channels in cell physiology is regulated by processes occurring after protein biosynthesis, which are critical for both channel function and targeting of channels to appropriate cell compartments. Here we apply biochemical and electrophysiological methods to investigate the role of the high-conductance, calcium-activated potassium (Maxi-K) channel C-terminal domain in channel tetramerization, association with the beta1 subunit, trafficking of the channel complex to the cell surface, and channel function. No evidence for channel tetramerization, cell surface expression, or function was observed with Maxi-K(1)(-)(323), a construct truncated three residues after the S(6) transmembrane domain. However, Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) are able to form tetramers and to associate with the beta1 subunit. Maxi-K(1)(-)(343)-beta1 and Maxi-K(1)(-)(441)-beta1 complexes are efficiently targeted to the cell surface and cannot be pharmacologically distinguished from full-length channels in binding experiments but do not form functional channels. Maxi-K(1)(-)(651) forms tetramers and associates with beta1; however, the complex is not present at the cell surface, but is retained intracellularly. Maxi-K(1)(-)(651) surface expression and channel function can be fully rescued after coexpression with its C-terminal complement, Maxi-K(652)(-)(1113). However coexpression of Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) with their respective C-terminal complements did not rescue channel function. Together, these data demonstrate that the domain(s) in the Maxi-K channel necessary for formation of tetramers, coassembly with the beta1 subunit, and cell surface expression resides within the S(0)-S(6) linker domain of the protein, and that structural constraints within the gating ring in the C-terminal region can regulate trafficking and function of constructs truncated in this region.

Androgens produce acute vasodilation of systemic, pulmonary, and coronary arteries in several mammal preparations and increase cardiomyocyte contractility. A decrease of the spontaneous beating of sinoatrial cells has also been described. The aim of this study was to characterize the direct effect of 5alpha-dihydrotestosterone on the spontaneous chronotropism and inotropism in the same preparation as an approach to establish the effect on cardiac output and their mechanism of action. The effects were studied on isolated right atria of Wistar rats placed in an organ bath in Tyrode solution at 37 degrees C and bubbled with carbogen. In male rats, the acute administration of 5alpha-dihydrotestosterone, a nonaromatizable derivate of testosterone, elicited a positive inotropism, which was associated with a negative chronotropism. As reported in the left atria, polyamines and beta-adrenoceptors played a role in 5alpha-dihydrotestosterone-elicited positive inotropism because the effect was antagonized by alpha-difluoromethylornithine, an inhibitor of polyamine synthesis, and atenolol, a beta1-adrenoceptor blocker, but not on the negative effect on chronotropism. The androgen increased the sinoatrial node recovery time, suggesting an effect on the mechanisms of spontaneous diastolic depolarization involved in atria pacemaking. These effects of 5alpha-dihydrotestosterone are not hormonally regulated because they are similarly produced in estrogenized females and gonadectomized male and female rats. These results suggest that the androgen could acutely improve cardiac performance.

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Functional and biochemical studies were performed in isolated left atria of male Wistar rats to study whether endogenous polyamines may mediate androgen-elicited positive inotropism and their relationship with a rise in cAMP during the cardiotonic effect. 5 alpha-Dihydrotestosterone (100 microM) exposure increased intracellular putrescine as determined by HPLC, but it did not increase spermidine and spermine. This effect was antagonized by an inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine (10 mM), suggesting enzyme activation. alpha-Difluoromethylornithine also antagonized androgens-elicited inotropism and the increase in intracellular cAMP. Putrescine (1 to 10 mM) elicited a concentration-dependent positive inotropism associated with the cAMP increase. The prior incubation with putrescine antagonized 5 alpha-dihydrotestosterone-elicited inotropism and did not produce sinergism on intracellular cAMP. Short-term incubation with 5 alpha-dihydrotestosterone or forskolin shifted to the left the cardiotonic effect of isoproterenol, an agonist of beta-adrenoceptors, without any increase in Emax, suggesting that a common mechanism was involved. Therefore, polyamines might modulate the cAMP production associated with the cardiotonic effect of androgens.