Calcium-sensitive inactivation in the gating of single calcium channels.

Voltage-activated calcium channels open and close, or gate, according to molecular transition rates that are regulated by transmembrane voltage and neurotransmitters. Here evidence for the control of gating by calcium was found in electrophysiological records of single, L-type calcium channels in heart cells. Conditional open probability analysis revealed that calcium entry during the opening of a single channel produces alterations in gating transition rates that evolve over the course of hundreds of milliseconds. Such alteration of calcium-channel gating by entry of a favored permeant ion provides a mechanism for the short-term modulation of single-ion channels.

Submicroscopic Ca2+ diffusion mediates inhibitory coupling between individual Ca2+ channels.

Dihydropyridine-sensitive Ca2+ channels in heart demonstrate an important negative feedback property: they close, or inactivate, in response to prior Ca2+ entry. We now find that Ca2+ influx through one channel can selectively contribute to the inactivation of another adjacent channel, without a generalized elevation of bulk intracellular Ca2+ concentration. Intracellular application of the Ca2+ chelator BAPTA greatly diminishes such negative interactions within Ca2+ channel pairs. These findings demonstrate that Ca2+ currents are controlled not only by intrinsic channel properties, but also by local diffusive interactions among neighboring channels. Such inhibitory coupling among channels provides a concrete example of localized Ca2+ signaling, long proposed to exist on the basis of theoretical calculations.

Mechanism of Ca(2+)-sensitive inactivation of L-type Ca2+ channels.

Many high threshold, voltage-gated Ca2+ channels, including the dihydropyridine-sensitive class (L-type), inactivate in response not only to voltage, but also to entry of Ca2+. Despite the physiological importance of this Ca(2+)-sensitive inactivation, its molecular mechanism is understood only in broad outline. We now demonstrate that Ca(2+)-dependent inactivation transpires by a Ca(2+)-induced shift of channel gating to a low open probability mode, distinguished by a more than 100-fold reduction of entry rate to the open state. A gating mechanism that explains this shift quantitatively and enables successful separation of Ca(2+)- and voltage-sensitive forms of inactivation is deduced and tested. Finally, both calmodulin activation and channel (de)phosphorylation are excluded as significant signaling events underlying Ca(2+)-induced mode shifts, leaving direct binding of Ca2+ to the channel as a likely chemical initiation event for inactivation.

Energetic and structural interactions between delta-dendrotoxin and a voltage-gated potassium channel.

Dendrotoxin proteins isolated from Mamba snake venom block potassium channels with a high degree of specificity and selectivity. Using site-directed mutagenesis we have identified residues that constitute the functional interaction surfaces of delta-dendrotoxin and its voltage-gated potassium channel receptor. delta-Dendrotoxin uses a triangular patch formed by seven side-chains (Lys3, Tyr4, Lys6, Leu7, Pro8, Arg10, Lys26) to block K(+) currents carried by a Shaker potassium channel variant. The inhibitory surface of the toxin interacts with channel residues at Shaker positions 423, 425, 427, 431, and 449 near the pore. Amino acid mutations that interact across the toxin-channel interface were identified by mutant cycle analysis. These results constrain the possible orientation of dendrotoxin with respect to the K(+) channel structure. We propose that dendrotoxin binds near the pore entryway but does not act as a physical plug.

A snake toxin inhibitor of inward rectifier potassium channel ROMK1.

Mamba snake dendrotoxins have been used extensively in biochemical and physiological studies of K+ channels of the brain. Their known targets of inhibition have been limited to the family of voltage-gated K+ channels. We report the isolation of a dendrotoxin inhibitor of ROMK1, a channel belonging to the inward rectifier family of K+ channels. The inhibitory activity, fractionated to purity with FPLC and HPLC, is identical to a previously identified delta-dendrotoxin. To verify that delta-dendrotoxin blocks ROMK1 channels, a cDNA encoding the toxin was synthesized and recombinant toxin expressed in Escherichia coli. Electrophysiological recordings reveal that recombinant delta-dendrotoxin has a half-maximal inhibition constant (Kd) of 150 nM when applied to ROMK1 channels expressed in Xenopus laevis oocytes. That the delta-dendrotoxin binding site exists on separate K+ channel classes is shown by its high affinity for two of the voltage-gated family of channels, Kv1.1 (Kd < 0.1 nM) and Kv1.6 (Kd = 23 nM). Single amino acid substitutions in ROMK1 indicate that delta-dendrotoxin binds to the pore region of ROMK1 even though it does not completely block conduction through the pore. These results suggest that dendrotoxins inhibit K+ channels by recognizing the structurally conserved pore region of these channels.

Characterization of recombinant rabbit cardiac and skeletal muscle Ca2+ release channels (ryanodine receptors) with a novel [3H]ryanodine binding assay.

A rapid assay for high affinity [3H]ryanodine binding to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized recombinant or native Ca2+ release channel proteins (ryanodine receptor, RyR) was devised. The key to preservation of high affinity [3H]ryanodine binding sites in the presence of increasing concentrations of CHAPS was the addition of phosphatidylcholine. This assay was used to characterize the equilibrium and kinetic properties of [3H]ryanodine binding to recombinant skeletal (RyR1) and cardiac (RyR2) Ca2+ release channels and the effects on binding of physiological modulators including ATP, Ca2+, and Mg2+. Both RyR1 and RyR2 had a single high affinity ryanodine binding site and low affinity sites, but [3H]ryanodine binding to recombinant RyR2 was not sensitive to ATP activation or Ca2+ inactivation and was less sensitive to Mg2+ inhibition. The [3H]ryanodine binding assay was used to estimate the expression level of recombinant RyR2 and RyR1, and to show that RyR2 can be expressed at very high levels in HEK-293 cells. Analysis of the properties of recombinant RyR2 and RyR1 by measurement of intracellular Fura-2 fluorescence revealed that the different properties of RyR2 and RyR1 are retained in the recombinant expressed proteins.

Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor).

We report transient expression of a full-length cDNA encoding the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (ryanodine receptor) in HEK-293 cells. The single-channel properties of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate-solubilized and sucrose gradient-purified recombinant Ca2+ release channels were investigated by using single-channel recordings in planar lipid bilayers. The recombinant Ca2+ release channel exhibited a K+ conductance of 780 pS when symmetrical 250 mM KCl was used as the conducting ion and a Ca2+ conductance of 116 pS in 50 mM luminal Ca2+. Opening events of the recombinant channels were brief, with an open time constant of approximately 0.22 ms. The recombinant Ca2+ release channel was more permeable to Ca2+ than to K+, with a pCa2+/pK+ ratio of 6.8. The response of the recombinant Ca2+ release channel to various concentrations of Ca2+ was biphasic, with the channel being activated by micromolar Ca2+ and inhibited by millimolar Ca2+. The recombinant channels were activated by ATP and caffeine, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Most recombinant channels were asymmetrically blocked, conducting current unidirectionally from the luminal to the cytoplasmic side of the channel. These data demonstrate that the properties of recombinant Ca2+ release channel expressed in HEK-293 cells are very similar, if not identical, to those of the native channel.