Toward a Multipathway Perspective: pH-Dependent Kinetic Selection of Competing Pathways and the Role of the Internal Glutamate in Cl-/H+ Antiporters

Abstract

Canonical descriptions of multistep biomolecular transformations generally follow a single-pathway viewpoint, with a series of transitions through intermediates converting reactants to products or repeating a conformational cycle. However, mounting evidence suggests that more complexity and pathway heterogeneity are mechanistically relevant due to the statistical distribution of multiple interconnected rate processes. Making sense of such pathway complexity remains a significant challenge. To better understand the role and relevance of pathway heterogeneity, we herein probe the chemical reaction network of a Cl^-/H⁺ antiporter, ClC-ec1, and analyze reaction pathways using multiscale kinetic modeling (MKM). This approach allows us to describe the nature of the competing pathways and how they change as a function of pH. We reveal that although pH-dependent Cl^-/H⁺ transport rates are largely regulated by the charge state of amino acid E148, the charge state of E203 determines relative contributions from coexisting pathways and can shift the flux pH-dependence. The selection of pathways via E203 explains how ionizable mutations (D/H/K/R) would impact the ClC-ec1 bioactivity from a kinetic perspective and lends further support to the indispensability of an internal glutamate in ClC antiporters. Our results demonstrate how quantifying the kinetic selection of competing pathways under varying conditions leads to a deeper understanding of the Cl^-/H⁺ exchange mechanism and can suggest new approaches for mechanistic control.

Figure 1.. ClC-ec1 and multiscale kinetic modeling (MKM) representation.

(A) ClC-ec1 structure (PDB ID: 1OTS) showing two monomers (ribbon representation), essential residues E148 and E203 (sticks), crystallographic Cl⁻ ions (green spheres), and outer Cl⁻ binding site, S_out, which is not occupied in 1OTS (dashed circles). Ion pathways are indicated by dashed arrows (dark green for Cl⁻, red for H⁺). Only the biological orientation is depicted (E148 on the extracellular side, E203 on the intracellular side). (B) Six descriptors used by MKM to model Cl⁻/H⁺ exchange: Cl⁻ binding to S_int, S_cen, or S_out; charge states of E148 and E203; and side-chain orientation (“up” or “down”) of E148. Each descriptor can be “0” (Cl⁻ absent, Glu deprotonated, E148 down) or “1” (Cl⁻ present, Glu protonated, E148 up). Every system state can thus be described by a unique six-digit binary number. For example, state 100011 has a deprotonated up E148, a deprotonated E203, and two Cl⁻ ions bound to S_cen and S_int. (C) Schematic of a H⁺ influx pathway comprised of five system states. Each state is indexed by a unique decimal number (e.g., 35) converted from the corresponding binary number (e.g., 100011). Note that the pathway has no net Cl⁻ transfer.

Figure 2.. Illustration of a pathway diagram and the associated pH-dependent flux profiles.

(A) Cl⁻/H⁺ pathways from the MKM solution set #1 in biological orientation reported in ref . H⁺ pathway (left) is indicated by orange arrows while two Cl⁻ pathways (right) are indicated by dark green and blue arrow, respectively. The width of an arrow represents the transition rate with the RLS highlighted by an asterisk. Latin numerals mark the transitions along the H⁺ pathway. (B) pH-dependent fluxes for H⁺ pathway. (C) pH-dependent fluxes for Cl⁻ pathways. The associated lines are the best fit to either eq 1 or bell-shaped distribution. The colors of the flux profiles are consistent with the representing pathway arrows. Note that the H⁺ transfer pathway has no net Cl⁻ flux and vice versa.

Figure 3.. Pathway heterogeneity for H⁺ transport in biological orientation.

Schematic representation and associated pH-dependent fluxes of single (AB), parallel (CD) and competing (EF) H⁺ transport pathways. The images and numbers display the ClC-ec1 states. The width of an arrow represents the transition rate with the RLS highlighted by an asterisk. Lines associated with fluxes are the best fit to eq 1 or a bell-shaped distribution. Note that there is no net Cl⁻ flux along H⁺ transfer pathways and although each transition is reversible only the flux-relevant directions are shown.

Figure 4.. Pathway heterogeneity in Cl⁻ transport in biological orientation.

(A) Schematic representation of major (I) and minor (II) Cl⁻ microscopic pathways. The images and numbers display the ClC-ec1 states. The width of an arrow represents the transition rate with the RLS highlighted by an asterisk. pH-dependent Cl⁻ fluxes through competitive pathways with a low (B) and high (C) E203 pK_a. Lines associated with fluxes are the best fit to eq 1 or a bell-shaped distribution. Note that there is no net H⁺ flux along Cl⁻ transfer pathways, and although each transition is reversible, only the flux-relevant directions are shown.