Purpose: There are conflicting studies of the impact of androgen deprivation therapy (ADT) on cardiovascular (CV) mortality among prostate cancer patients receiving curative intent external beam radiation therapy (EBRT). We assessed the impact of ADT on CV mortality in patients treated in British Columbia.Methods and Materials: Provincial pharmacy and radiotherapy databases were linked to the provincial cancer registry, and defined a cohort of patients treated with curative intent EBRT between 1998 and 2005. We determined the duration of ADT and the cumulative incidence of CV death. We compared death from CV disease with and without ADT, and by duration of ADT using competing risk analysis and Fine and Gray multivariant analysis. A total of 600 randomly selected patients were reviewed to determine baseline CV disease, CV risk factors, and Charlson Index.Results: Of 5,948 prostate cancer patients treated with radical intent EBRT, of whom 1,933 were treated without ADT, 674 received ADT for <= 6 months and 3,341 received > 6 months of ADT. The cumulative CV mortality at 7 years was 2.6% (95% confidence interval [CI] 1.9-3.5%), 2.1% (95% CI = 1.2-3.5%), and 1.4(95% CI = 1.0-2.0%) for patients with no ADT, <= 6 months of ADT, and > 6 months of ADT, respectively (Gray's p = 0.002). Baseline CV disease and risk factors were more prevalent in the no-ADT group compared with the > 6-month ADT group.Conclusions: This study demonstrated a lower CV mortality rate among patients treated with longer durations of ADT than those treated without ADT. These differences likely relate to selection of patients for ADT rather than effect of ADT itself. (C) 2011 Elsevier Inc.

The N-termini of Kv1 α-subunits bind co-translationally with cytosolic Kvβ-subunits, with 1:1 symmetry. Kvβ-subunits of three distinct families have been found in neural and cardiac tissue, and members of the Kvβ1 family confer fast inactivation and slowed deactivation when co-assembled with Kv1 α-subunits. These effects may be due to a blocking action by the Kvβ1 N-terminus. Kvβ1 subunits also cause an apparent hyperpolarizing shift in the activation curve of Kv1 channels, which may be a consequence of block by the Kvβ1 N-terminus, due to premature saturation of deactivating tail currents, or alternately may be due to an allosteric interaction between Kvβ1 and Kv1 α-subunits, modifying voltage sensor movement. Here, we use voltage clamp fluorometry to directly track the movement of the Kv1.2 voltage sensor in the absence or presence of the Kvβ1.2 subunit, or an N-terminally-truncated Kvβ1.2 subunit which does not produce fast inactivation. While Kvβ1.2 led to a spike-and-decay current waveform and a hyperpolarized shift in ionic current activation, the voltage dependence of ON gating charge movements were unaffected. Kvβ1.2 also slowed Kv1.2 fluorescence and current deactivation, implying that the return of the voltage sensor to its pre-activation position followed the closing of the activation gate. These findings suggest that the hyperpolarizing shift in channel activation is a consequence of pore block by the Kvβ1.2 N-terminus, and not an allosteric effect on the Kv1.2 voltage sensor, and that block prevents both closure of the activation gate and the return of the S4 helix upon repolarization.

Fluorescence-based approaches provide powerful techniques to directly report structural dynamics underlying gating processes in Shaker K-V channels. Here, following on from work carried out in Shaker channels, we have used voltage clamp fluorimetry for the first time to study voltage sensor motions in mammalian K(V)1.5 channels, by attaching TMRM fluorescent probes to substituted cysteine residues in the S3-S4 linker of K(V)1.5 (A397C). Compared with the Shaker channel, there are significant differences in the fluorescence signals that occur on activation of the channel. In addition to a well-understood fluorescence quenching signal associated with S4 movement, we have recorded a unique partial recovery of fluorescence after the quenching that is attributable to gating events at the outer pore mouth, 1 that is not seen in Shaker despite significant homology between it and K(V)1.5 channels in the S5-P loop-S6 region. Extracellular potassium is known to modulate C-type inactivation in Shaker and K-V channels at sites in the outer pore mouth, and so here we have measured the concentration-dependence of potassium effects on the fluorescence recovery signals from A397C. Elevation of extracellular K+ inhibits the rapid fluorescence recovery, with complete abolition at 99 mM K+, and an IC50 of 29 mM K+ (o). These experiments suggest that the rapid fluorescence recovery reflects early gating movements associated with inactivation, modulated by extracellular K+, and further support the idea that outer pore motions occur rapidly after K(V)1.5 channel opening and can be observed by fluorophores attached to the S3-S4 linker.

Cytosolic K-V beta 1 subunits co-assemble with transmembrane K(V)1 channel alpha-subunits and have complex effects on channel function. Fast inactivation, the most obvious effect conferred, is due to fast open channel block resulting from the binding of the N-terminus within the inner mouth of the pore. K-V beta 1 subunits also slow current deactivation, enhance slow inactivation and shift channel activation to more negative voltages, but the mechanisms underlying these actions are not known. Here we use voltage clamp fluorimetry at sites near the extracellular end of the S4 helix, the channel's primary voltage sensor, in combination with voltage clamp electrophysiology, to independently track the movement of the S4 helix along with ionic current, and thus identify the structural and mechanistic means by which the K-V beta 1.2 subunit confers its actions on the K(V)1.2 channel. We show that the negative shift in current activation is not due to direct actions of K-V beta 1.2 on the S4 segment. Instead, this shift results from an apparent saturation of channel activation at depolarized potentials as the extent of open channel block by the K-V beta 1.2 N-terminus progressively increases. The return of fluorescence to baseline is slowed along with current deactivation. According to our data, this is due to an inability of the activation gate to close while the K-V beta 1.2 N-terminus occupies the pore and strong coupling of the gate with the S4 segment. Together with data from previous studies, our findings provide a complete and coherent picture of the functional and structural interactions between K-V beta 1.2 and K(V)1.2.

Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K+ ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3–S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 ± 3% of the total signal and occurred with a τ of 3.4 ± 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K+ to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.