The existence of a large number of receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) raises the question of how a particular receptor selectively regulates specific targets. We provide insight into this question by identifying a prototypical macromolecular signaling complex. The beta(2) adrenergic receptor was found to be directly associated with one of its ultimate effectors, the class C L-type calcium channel Ca(v)1.2. This complex also contained a G protein, an adenylyl cyclase, cyclic adenosine monophosphate-dependent protein kinase, and the counterbalancing phosphatase PP2A. Our electrophysiological recordings from hippocampal neurons demonstrate highly localized signal transduction from the receptor to the channel. The assembly of this signaling complex provides a mechanism that ensures specific and rapid signaling by a G protein-coupled receptor.

We investigated the internal pH-sensitivity of heterologously expressed hSlo1 BK channels. In the virtual absence of Ca(2+) and Mg(2+) to isolate the voltage-dependent gating transitions, low internal pH enhanced macroscopic hSlo1 currents by shifting the voltage-dependence of activation to more negative voltages. The activation time course was faster and the deactivation time course was slower with low pH. The estimated K(d) value of the stimulatory effect was approximately pH = 6.5 or 0.35 micro M. The stimulatory effect was maintained when the auxiliary subunit mouse beta1 was coexpressed. Treatment of the hSlo1 channel with the histidine modifying agent diethyl pyrocarbonate also enhanced the hSlo1 currents and greatly diminished the internal pH sensitivity, suggesting that diethyl pyrocarbonate and low pH may work on the same effector mechanism. High concentrations of Ca(2+) or Mg(2+) also masked the stimulatory effect of low internal pH. These results indicate that the acid-sensitivity of the Slo BK channel may involve the channel domain implicated in the divalent-dependent activation.

Oxidation of amino acid residues causes noticeable changes in gating of many ion channels. We found that P/C-type inactivation of Shaker potassium channels expressed in Xenopus oocytes is irreversibly accelerated by patch excision and that this effect was mimicked by application of the oxidant H(2)O(2), which is normally produced in cells by the dismutase action on the superoxide anion. The inactivation time course was also accelerated by high concentration of O(2). Substitution of a methionine residue located in the P-segment of the channel with a leucine largely eliminated the channel's sensitivity to patch excision, H(2)O(2), and high O(2). The results demonstrate that oxidation of methionine is an important regulator of P/C-type inactivation and that it may play a role in mediating the cellular responses to hypoxia/hyperoxia.

Dihydropyridines (DHPs) are well known for their effects on L-type voltage-dependent Ca2+ channels. However, these drugs also affect other voltage-dependent ion channels, including Shaker K+ channels. We examined the effects of DHPs on the Shaker K+ channels expressed in Xenopus oocytes. Intracellular applications of DHPs quickly and reversibly induced apparent inactivation in the Shaker K+ mutant channels with disrupted N- and C-type inactivation. We found that DHPs interact with the open state of the channel as evidenced by the decreased mean open time. The DHPs effects are voltage-dependent, becoming more effective with hyperpolarization. A model which involves binding of two DHP molecules to the channel is consistent with the results obtained in our experiments.

Drosophila genes reaper, grim, and headinvolution-defective (hid) induce apoptosis in several cellular contexts. N-terminal sequences of these proteins are highly conserved and are similar to N-terminal inactivation domains of voltage-gated potassium (K ؉ ) channels. Synthetic Reaper and Grim N terminus peptides induced fast inactivation of Shaker-type K ؉ channels when applied to the cytoplasmic side of the channel that was qualitatively similar to the inactivation produced by other K ؉ channel inactivation particles. Mutations that reduce the apoptotic activity of Reaper also reduced the synthetic peptide's ability to induce channel inactivation, indicating that K ؉ channel inactivation correlated with apoptotic activity. Coexpression of Reaper RNA or direct injection of full length Reaper protein caused near irreversible block of the K ؉ channels. These results suggest that Reaper and Grim may participate in initiating apoptosis by stably blocking K ؉ channels.