A number of potassium channels including members of the KCNQ family and the Ca 2+ activated IK and SK, but not BK, are strongly and reversibly regulated by small changes in cell volume. It has been argued that this general regulation is mediated through sensitivity to changes in membrane stretch. To test this hypothesis we have studied the regulation of KCNQ1 and BK channels after expression in Xenopus oocytes. Results from cell-attached patch clamp studies (∼50 μm 2 macropatches) in oocytes expressing BK channels demonstrate that the macroscopic volume-insensitive BK current increases with increasing negative hydrostatic pressure (suction) applied to the pipette. Thus, at a pipette pressure of −5.0 ± 0.1 mmHg the increase amounted to 381 ± 146% (mean ± s.e.m., n = 6, P < 0.025). In contrast, in oocytes expressing the strongly volume-sensitive KCNQ1 channel, the current was not affected by membrane stretch. The results indicate that (1) activation of BK channels by local membrane stretch is not mimicked by membrane stress induced by cell swelling, and (2) activation of KCNQ1 channels by cell volume increase is not mediated by local tension in the cell membrane. We conclude that stretch and volume sensitivity can be considered two independent regulatory mechanisms.
Xenopus oocytes release ATP in response to mechanical stimuli and cell volume changes. Purinergic P2 and P1 receptors confer some of the KCNQ1 channel volume sensitivity, although endogenous adenosine receptors and expressed P2Y2 receptors do so in the negative direction.
Association of the voltage‐activated potassium channel KCNQ1 with the accessory protein subunit KCNE1 gives rise to the cardiac IKs delayed rectifier potassium current. Aside from altering the kinetics of the KCNQ1 channel current, KCNE1 also augments the KCNQ1 current. This increase in macroscopic current is due either to an increase in ion channel conductance (γ), the open state probability (Po) or an increase in the number of channels in the plasma membrane (N). The latter can be quantified by measuring the level of KCNQ1 surface expression by using an enzyme‐linked immunoassay. To do this, we employed a HA‐tagged version of the KCNQ1 channel and expressed it in presence or absence of KCNE1 in Xenopus oocytes. The HA‐tag, which is located at the second extracellular loop of the protein, allowed us to “count” the number of KCNQ1 channels expressed in the cell membrane. In parallel, currents were measured with two electrode voltage clamp. The results show that the KCNQ1 surface expression is ~70% lower when KCNE1 is co‐expressed compared to KCNQ1 alone despite a ~20% higher current for the heteromeric KCNQ1/KCNE1. This indicates that the overall increase of the KCNQ1 current, when co‐expressed with KCNE1, is not due to an increase in ion channel surface expression but rather to an increase in single‐channel conductance or in open state probability.
Reactive oxygen species (ROS) play an important role in the progressive neuronal function loss that is part of both the normal ageing process and neurodegenerative disease. A central question is whether voltage-gated K þ (Kv) channels, which are key regulators of neuronal excitability, are physiological targets of ROS and whether these interactions have a role in the mechanisms underlying age-related neurodegeneration. Here, we show that oxidation of K þ channel KVS-1 during ageing causes sensory function loss in Caenorhabditis elegans, and that protection of this channel from oxidation preserves neuronal function.-Thus, chemotaxis to biotin and lysine, a function controlled by KVS-1, was significantly impaired (70%) in normal or wild-type young worms exposed to chloramine-T (CHT) or hydrogen peroxide (H 2 O 2 ), but only moderately affected (35%) in worms harboring an oxidation-reduction (redox)-resistant KVS-1 mutant (C113S). In ageing C113S worms, the effects of free radical accumulation were significantly attenuated (40% loss-of-function) compared to wild-type (75%). Electrophysiological analyses showed that both ROS accumulation during ageing, and acute exposure to oxidizing agents, acted primarily to modify native KVS-1 channels expressed in the ASER neuron (which mediates chemotaxis) and as a consequence altered the excitability of neurons harboring wild-type but not C113S KVS-1. Together, these findings establish a pivotal role for ROS-mediated oxidation of voltage-gated K þ channels in sensorial decline during ageing.
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