Caβ retains its ability to shift GV curves, but maximal conductances are reduced in channels bearing mutations of AID-exposed residues

(A) Macroscopic currents from oocytes coexpressing Caβ with either Ca1.2 E462R or Ca1.2 K465N during the same stimulation protocol used in (shown at the top), with calibration bars corresponding to 20 ms and 200 nA. (B) GV curves in the presence (filled symbol) or absence (open symbol) of Caβ. (C) Plots of tail current amplitudes normalized by Q (I/Q) for Ca1.2 WT (▪), Ca1.2 E462R (•), and Ca1.2 K465N (▴). I/Q (mean ± SEM) versus voltage plots were fitted to the sum of two Boltzmann distributions. The maximal I/Q was 17.8 ± 2.5 nA/pC ( = 24) for Ca1.2 WT + Caβ, 7.4 ± 1.74 nA/pC ( = 11) for Ca1.2 K465N + Caβ, and 3.5 ± 0.8 nA/pC pC ( = 10) for Ca1.2 E462R + Caβ.<p><b>Copyright information:</b></p><p>Taken from "Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation"</p><p></p><p>The Journal of General Physiology 2008;132(3):383-395.</p><p>Published online Jan 2008</p><p>PMCID:PMC2518731.</p><p></p

Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation-4

I/Q versus voltage from oocytes expressing Ca1.2 E462R after the injection of purified Caβ protein at the indicated concentrations. Traces correspond to superimposed responses to three 60-ms depolarizing pulses to −30 mV, 0 mV, and +30 mV from a holding voltage of −90 mV. Calibration bars correspond to 20 ms and 200 nA. Experimental I/Q values (8) were fitted to the equation (blue line):Each member of the equation corresponds to templates in absence (−) or presence (+) of saturating concentration of Caβ protein (2.0 μM). Variables defining each template were obtained from the fit to average I/Q plot from each condition. The contribution of +β and −β templates are shown as green and red lines, respectively. (B) As A) but for Ca1.2 K465N. (C) Mean ± SE of β2a-like versus protein concentration ([Caβ]) in μM. Continuous lines show the fit to a standard Hill equation:Where is the apparent dissociation constant and is the Hill coefficient. ranged between 1.4 and 1.6, whereas for WT, E462R, and K465N was 0.20, 0.22, and 0.25 μM, respectively. The number of averaged experiments ranged from three to six for every concentration and calcium channel variant.<p><b>Copyright information:</b></p><p>Taken from "Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation"</p><p></p><p>The Journal of General Physiology 2008;132(3):383-395.</p><p>Published online Jan 2008</p><p>PMCID:PMC2518731.</p><p></p

Single channel mean currents and I/Q plots from macroscopic currents from different Ca1

2 variants recorded in high Ba and S(-)Bay K8644. (A) Mean current traces for six patches containing single Ca1.2 WT/Caβ channels (black) from seven patches with Ca1.2 E462R/Caβ channels (blue), and from 6 with Ca1.2 K465N/Caβ channels (red). The number of traces averaged in each case was 4,032 for Ca1.2 WT/Caβ, 7,504 for Ca1.2 E462R/Caβ, and 6,104 for Ca1.2 K465N/Caβ. Voltage protocol and recording condition were as described in . Calibration bars correspond to 50 ms and 100 fA. (B) I/Q versus voltage plot for Ca1.2 WT ( = 12), Ca1.2 E462R ( = 12), and Ca1.2 K465N ( = 13) coexpressed with Caβ and recorded in external 76 mM Ba and 0.1 μM of S(-) Bay K 8644 as used for single channel.<p><b>Copyright information:</b></p><p>Taken from "Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation"</p><p></p><p>The Journal of General Physiology 2008;132(3):383-395.</p><p>Published online Jan 2008</p><p>PMCID:PMC2518731.</p><p></p

Deletion of AID sequence or replacement of the conserved tryptophan abolished modulation by Caβ

(A) Superimposed macroscopic current traces from oocytes coexpressing Caβ either with Ca1.2 W470S or Ca1.2 ΔAID. Each trace was obtained during a 70-ms pulse of increasing amplitude, starting at −40 mV and ending at 150 mV in 10-mV increments. Membrane was held at −80 mV until the beginning of the pulse and returned to −40 mV for the remaining of the trace (shown at the top). Currents were sampled at 2.5 kHz until 3 ms before the end of the pulse, and then at 50 kHz. Traces were filtered at 10 kHz, and a P/−4 prepulse protocol was used to subtract linear components. Calibration bars correspond to 20 ms and 200 nA. (B) Conductance–voltage relationship (GV curve) for the different subunit combinations shown in A. The peak amplitude of the tail currents for each test voltage was normalized by the largest tail current (I/Imax) to generate the GV curves. Open and filled symbols correspond to oocytes recorded with or without Caβ, respectively. The sums of two Boltzmann distributions that best described each set of data are shown as continuous lines.<p><b>Copyright information:</b></p><p>Taken from "Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation"</p><p></p><p>The Journal of General Physiology 2008;132(3):383-395.</p><p>Published online Jan 2008</p><p>PMCID:PMC2518731.</p><p></p

Functional Role of Solvent Entropy and Conformational Entropy of Metal Binding in a Dynamically Driven Allosteric System

Allostery is a regulatory phenomenon whereby ligand binding to one site influences the binding of the same or a different ligand to another site on a macromolecule. The physical origins of allosteric regulation remain under intense investigation. In general terms, ligand-induced structural changes, perturbations of residue-specific dynamics, and surrounding solvent molecules all potentially contribute to the global energetics of allostery. While the role of solvent is generally well understood in regulatory events associated with major protein structural rearrangements, the degree to which protein dynamics impact solvent degrees of freedom is unclear, particularly in cases of dynamically driven allostery. With the aid of new crystal structures, extensive calorimetric and residue-specific dynamics studies over a range of time scales and temperatures, we dissect for the first time the relative degree to which changes in solvent entropy and residue-specific dynamics impact dynamically driven, allosteric inhibition of DNA binding by Zn in the zinc efflux repressor, CzrA (chromosomal zinc-regulated repressor). We show that non-native residue-specific dynamics in allosterically impaired CzrA mutants are accompanied by significant perturbations in solvent entropy that cannot be predicted from crystal structures. We conclude that functional dynamics are not necessarily restricted to protein residues but involve surface water molecules that may be responding to ligand (Zn)-mediated perturbations in protein internal motions that define the conformational ensemble, rather than major structural rearrangements