DiFrancesco ML et al. (2014), Effect of cytosolic pH on inward currents revea...

XB-ART-49418

PLoS One 2014 Jan 01;99:e107406. doi: 10.1371/journal.pone.0107406.

Show Gene links Show Anatomy links

Effect of cytosolic pH on inward currents reveals structural characteristics of the proton transport cycle in the influenza A protein M2 in cell-free membrane patches of Xenopus oocytes.

DiFrancesco ML , Hansen UP , Thiel G , Moroni A , Schroeder I .

???displayArticle.abstract???
Transport activity through the mutant D44A of the M2 proton channel from influenza virus A was measured in excised inside-out macro-patches of Xenopus laevis oocytes at cytosolic pH values of 5.5, 7.5 and 8.2. The current-voltage relationships reveal some peculiarities: 1. "Transinhibition", i.e., instead of an increase of unidirectional outward current with increasing cytosolic H(+) concentration, a decrease of unidirectional inward current was found. 2. Strong inward rectification. 3. Exponential rise of current with negative potentials. In order to interpret these findings in molecular terms, different kinetic models have been tested. The transinhibition basically results from a strong binding of H(+) to a site in the pore, presumably His37. This assumption alone already provides inward rectification and exponential rise of the IV curves. However, it results in poor global fits of the IV curves, i.e., good fits were only obtained for cytosolic pH of 8.2, but not for 7.5. Assuming an additional transport step as e.g. caused by a constriction zone at Val27 resulted in a negligible improvement. In contrast, good global fits for cytosolic pH of 7.5 and 8.2 were immediately obtained with a cyclic model. A "recycling step" implies that the protein undergoes conformational changes (assigned to Trp41 and Val27) during transport which have to be reset before the next proton can be transported. The global fit failed at the low currents at pHcyt = 5.5, as expected from the interference of putative transport of other ions besides H(+). Alternatively, a regulatory effect of acidic cytosolic pH may be assumed which strongly modifies the rate constants of the transport cycle.

???displayArticle.pubmedLink??? 25211283
???displayArticle.pmcLink??? PMC4174909
???displayArticle.link??? PLoS One

Species referenced: Xenopus laevis
Genes referenced: kmo naa50

???attribute.lit??? ???displayArticles.show???

	Figure 2. Activation of M2D44A by external acidification.Representative currents obtained from water- or M2D44A-injected Xenopus oocytes were recorded at â20 mV in TEVC. External pH was changed and amantadine added as a blocker as indicated by the bars. The internal pH was that of the oocyte, about pH 7.4.
	Figure 3. Dependence of currents on cytosolic pH in cell-free macro-patches.Representative traces are shown from (A) water-injected oocytes and (B) M2D44A-injected oocytes. The pH values are given at the traces. The averaged IV curves from patches clamped to test voltages between â100 mV to +100 mV are shown in (C) for H2Oâ and (D) for M2D44A-injected oocytes. The current traces at high negative membrane potentials (B) were noisy. However, the IV curves obtained after averaging show a smooth behavior at high negative potentials (D). The individual data is given in Table S1 in file S2.
	Figure 4. Averaged IV curves of M2D44A obtained from macro-patches at different cytosolic pH: 5.5 (squares), 7.5 (circles) and 8.2 (triangles) (nâ=â2, 3, 3, respectively).The error bars give the standard deviation of the measured data points. The external pH was 5.5. Averaged IV curves from water-injected oocytes (different pH values were measured on each control oocyte) have been subtracted from the original IV curves measured in patches expressing M2D44A (nâ=â5â7 per pH value) in order to isolate the M2 current from the endogenous currents. The data for pHcytâ=â7.5 and 8.2 was fitted with the linear model with a strongly binding residue (enzyme model) in the pore (Eqs. 2 and 3). Voltage acts on the B-O transitions (Eqs. 4b, c). Cytosolic H+ binds to the U-B transition kCB (Eq. 4a). Since the fit is not perfect, in particular for the data at pH 7.5, the full equations used for fitting are not given here, but in Eq. S4 in file S1).
	Figure 5. Reaction schemes for the dependence of proton transport in M2D44A on cytosolic pH.(A) Cyclic reaction with the following states of the M2 protein. O: the external part of the protein has taken up a proton from the external side or is ready to release a proton to the external solution. B: the protein has bound a proton from O via the voltage-sensitive translocation kOB or from C (kCB) and is ready for the voltage-sensitive translocation of this proton to O (kBO) or release to C (kBC), C: the protein is ready to bind a cytosolic proton and transfer it to B with the rate constant kCB or to take a proton from B (kBC) and release it to the internal side. The recycling step O-C includes the resetting of the conformational changes of the protein necessary for the transport of the next proton. It also includes the exchange of a proton with the external solution. (B) Augmented model derived from the structural information as detailed in the Introduction. The meaning of the symbols is as follows: Capital letter: Tâ=âTrp41-basket; Vâ=âVal27-gate, Pâ=âpool in the cavity filled with m or n protons, m>n; small letter oâ=âopen, iâ=âopen, but instable câ=âclosed; Number: Protonation state of the His37-box. The equality sign indicates which state of the augmented model is assigned to a state in the model in (A). The solid boxes include reactions, which are merged to gross reactions in (A) as explained in file S1. The dotted box indicates the gross reaction kMC and kCM used in the recycling branch of the model in (C). (C) The recycling pathway with the gross rate constants ÎºOC and ÎºCO of (A) is split up into two steps: O-M with external proton binding kMO (and release kOM), and recycling (M-C) with the gross rate constants kMC and kCM as indicated by the dotted box in (B).
	Figure 6. Fit of the data of Fig. 4 with the cyclic reaction scheme.The lines give the fits by means of Eqs. S13âS15 in file S1. For pH 5.5, two fits are shown: red curve: joint fit with curves for cytosolic pH of 7.5 and 8.2, black curve: without the restriction by the joint fit. This is illustrated by two different âfreeâ fits at pH 5.5 (Table 1, data columns 4 and 5), which show that different sets of rate constants can fit the curve.
	Figure 7. Properties of the M2D44A transport cycle as calculated from the cyclic model in Fig. 5A.(A) Occupation probabilities of the three states in the model and their dependence on membrane potential V and cytosolic pHcyt. (B) Unidirectional currents Iinâ=ââf O kOB (red) and Ioutâ=âf B kBO (blue, pH 7.5 and 8.2 coincide) and their dependence of membrane potential V and cytosolic pHcyt. The inset shows the unidirectional outward currents on an expanded scale.
	Figure 8. Calculated IV curves for different external pH of 5.5, 6.5 and 7.5.The parameters in Table 1 from the joint fit for pHcytâ=â8.2 with ÎºCO modified by pHext (Eq. 6) were used to generate the curves; pHext is given at the curves. For comparison, triangles show the experimental data of Fig. 6 for pHcytâ=â8.2/pHextâ=â5.5.
	Figure 1. Structure of the M2 protein and its orientation during infection.Coordinates were taken from [15], pbd access code 3lbw. The image was created with the UCSF Chimera package [67], which is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311). In the present experimental setting, in which the M2 protein is expressed in Xenopus oocytes, the endosomal side corresponds to the external medium and the side facing to the interior of the virus particle corresponds to the â€œcytosolicâ€ side. The critical residues Val27 (green), His37 (blue) and Trp41 (red) are shown explicitly.

References [+] :

Abenavoli, Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv. 2009, Pubmed, Xenbase