Proton probing of the charybdotoxin binding site of Shaker K+ channels.

We have investigated the interaction of charybdotoxin (CTX) with Shaker K channels. We substituted a histidine residue for the wild-type phenylalanine (at position 425) in an inactivation-removed channel. The nature of the imidazole ring of the histidine provides the ability to change the charge on this amino acid side chain with solution hydrogen ion concentration. Wild-type, recombinant CTX blocked wild-type Shaker channels in a bimolecular fashion with a half-blocking concentration (Kd) of 650 nM (at a membrane potential of 0 mV). The F425H mutant channels were much more sensitive to CTX block with an apparent Kd (at pH 7.0) of 75 nM. Block of F425H but not wild-type channels was strongly pH sensitive. A pH change from 7 to 5.5 rendered the F425H channels >200-fold less sensitive to CTX. The pH dependence of CTX block was steeper than expected for inhibition produced by H+ ions binding to identical, independent sites. The data were consistent with H+ ions interacting with subunits of the channel homotetrameric structure. The in situ pK for the imidazole group on the histidine at channel position 425 was determined to be near 6.4 and the dissociation constant for binding of toxin to the unprotonated channel was near 50 nM. We estimate that the binding of a H+ ion to each subunit adds 0.8 kcal/mol or more of interaction energy with CTX. We used mutant toxins to test electrostatic and steric interactions between specific CTX residues and channel position 425. Our results are consistent with a model in which protons on F425H channel subunits interact with three positive charges on CTX at an effective distance 6-7 A from this channel position.

Nonindependent K+ movement through the pore in IRK1 potassium channels.

We measured unidirectional K+ in- and efflux through an inward rectifier K channel (IRK1) expressed in Xenopus oocytes. The ratio of these unidirectional fluxes differed significantly from expectations based on independent ion movement. In an extracellular solution with a K+ concentration of 25 mM, the data were described by a Ussing flux-ratio exponent, n', of approximately 2.2 and was constant over a voltage range from -50 to -25 mV. This result indicates that the pore of IRK1 channels may be simultaneously occupied by at least three ions. The IRK1 n' value of 2.2 is significantly smaller than the value of 3.5 obtained for Shaker K channels under identical conditions. To determine if other permeation properties that reflect multi-ion behavior differed between these two channel types, we measured the conductance (at 0 mV) of single IRK1 channels as a function of symmetrical K+ concentration. The conductance could be fit by a saturating hyperbola with a half-saturation K+ activity of 40 mM, substantially less than the reported value of 300 mM for Shaker K channels. We investigated the ability of simple permeation models based on absolute reaction rate theory to simulate IRK1 current-voltage, conductance, and flux-ratio data. Certain classes of four-barrier, three-site permeation models are inconsistent with the data, but models with high lateral barriers and a deep central well were able to account for the flux-ratio and single channel data. We conclude that while the pore in IRK1 and Shaker channels share important similarities, including K+ selectivity and multi-ion occupancy, they differ in other properties, including the sensitivity of pore conductance to K+ concentration, and may differ in the number of K+ ions that can simultaneously occupy the pore: IRK1 channels may contain three ions, but the pore in Shaker channels can accommodate four or more ions.

Autonomic modulation of action potential and tension in guinea pig papillary muscles.

The effects of alpha 1-adrenoceptor and muscarinic acetylcholine receptor stimulation on action potential and tension were studied in guinea pig papillary muscles obtained from both right and left ventricles. Stimulation of muscarinic acetylcholine receptors with carbachol produced a reduction of the action potential duration and a positive inotropic effect in papillary muscles from both ventricles. Both effects were concentration dependent and atropine sensitive. However, differential responsiveness was found upon alpha 1-adrenoceptor activation in muscles obtained from left and right ventricles. In right side papillary muscles, the alpha 1-adrenoceptor agonist, methoxamine, decreased the action potential duration and produced a positive inotropic effect. In contrast, methoxamine decreased the action potential duration but failed to produce a positive inotropic effect in left side papillary muscles. All methoxamine effects were antagonized by prazosin. Responses to maximum concentration of carbachol and methoxamine on the action potential duration and contractility were additive in right side papillary muscles. Phorbol 12,13-dibutyrate (PDB), a direct protein kinase C activator, also decreased the action potential duration in a manner that was additive to both carbachol and methoxamine. However, PDB reversed the positive inotropic effect of carbachol and methoxamine. The methoxamine-induced shortening of the action potential duration was prevented by pretreatment with indomethacin and nordihydroguaiaretic acid, blockers of arachidonic acid metabolism, but not by the protein kinase C antagonist, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7).(ABSTRACT TRUNCATED AT 250 WORDS)

H+ ion modulation of C-type inactivation of Shaker K+ channels.

The participation of an extracellular loop in C-type inactivation of voltage-gated K+ channels was investigated. A wild-type phenylalanine (at position 425) between the fifth putative transmembrane segment (S5) and the pore region of the Shaker K+ channel was mutated to a histidine and the functional consequences of protonating the imidizole group of the histidine were examined. C-type inactivation of both wild-type and mutant channels was sensitive to external pH over the range of 5.2-8. The pH dependence of wild-type channels was characterized by an apparent pK value of 5. 0. The inactivation kinetics of F425H mutant channels had a pH dependence with a pK of 5.8 - in addition to the pH dependence of the wild-type channels. Moreover, at pH 7 and 8 the voltage dependence of C-type inactivation kinetics was manifest only in the F425H mutant channels. C-type inactivation in wild-type channels involves a chemical group with a low pK. Taken together, these results suggest that residues located in the extracellular S5-pore loop of the Shaker K+ channel participate in C-type inactivation.

The multi-ion nature of the pore in Shaker K+ channels.

We have investigated some of the permeation properties of the pore in Shaker K channels. We determined the apparent permeability ratio of K+, Rb+, and NH4+ ions and block of the pore by external Cs+ ions. Shaker channels were expressed with the baculovirus/Sf9 expression system and the channel currents measured with the whole-cell variant of the patch clamp technique. The apparent permeability ratio, PRb/PK, determined in biionic conditions with internal K+, was a function of external Rb+ concentration. A large change in PRb/PK occurred with reversed ionic conditions (internal Rb+ and external K+). These changes in apparent permeability were not due to differences in membrane potential. With internal K+, PNH4/PK was not a function of external NH4+ concentration (at least over the range 50-120 mM). We also investigated block of the pore by external Cs+ ions. At a concentration of 20 mM, Cs+ block had a voltage dependence equivalent to that of an ion with a valence of 0.91; this increased to 1.3 at 40 mM Cs+. We show that a 4-barrier, 3-site permeation model can simulate these and many of the other known properties of ion permeation in Shaker channels.

Permeant anions control gating of calcium-dependent chloride channels.

The effects of external anions (SCN(-), NO3-, I(-), Br(-), F(-), glutamate, and aspartate) on gating of Ca(2+)-dependent Cl(-) channels from rat parotid acinar cells were studied using the whole-cell configuration of the patch-clamp technique. Shifts in the reversal potential of the current induced by replacement of external Cl(-) with foreign anions, gave the following selectivity sequence based on permeability ratios ( P(x)/ P(Cl)): SCN(-)>I(-)>NO3->Br(-)>Cl(-)>F(-)>aspartate>glutamate. Using a continuum electrostatic model we calculated that this lyotropic sequence resulted from the interaction between anions and a polarizable tunnel with an effective dielectric constant of approximately 23. Our data revealed that anions with P(x)/P(Cl) > 1 accelerated activation kinetics in a voltage-independent manner and slowed deactivation kinetics. Moreover, permeant anions enhanced whole-cell conductance ( g, an index of the apparent open probability) in a voltage-dependent manner, and shifted leftward the membrane potential- g curves. All of these effects were produced by the anions with an effectiveness that followed the selectivity sequence. To explain the effects of permeant anions on activation kinetics and g(Cl) we propose that there are 2 different anion-binding sites in the channel. One site is located outside the electrical field and controls channel activation kinetics, while a second site is located within the pore and controls whole-cell conductance. Thus, interactions of permeant anions with these two sites hinder the closing mechanism and stabilize the channel in the open state.