# Molecular Mechanisms of Ion Transport

> **NIH NIH R35** · NEW YORK UNIVERSITY SCHOOL OF MEDICINE · 2022 · $258,657

## Abstract

My laboratory is interested in fundamental molecular mechanisms by which cells maintain ionic homeostasis.
We study two particular systems responsible for transport of K+ and Zn2+, respectively. Overall goals are the
same for both systems, which are to develop a comprehensive understanding of transport from structural as
well as thermodynamic perspectives. We will use a broad spectrum of biophysical and biochemical
approaches, including cryo-EM for structure determination, in vitro biophysical assays for functional
characterization, single-molecule FRET and Molecular Dynamic simulations for analyzing dynamics of the
molecules. In this way, we aim to define an energy landscape for each system, annotated with the
experimental structures for stable intermediates as well as an appreciation for the high-energy transition states
that define the transport pathway. We also seek to understand determinants of substrate specificity and
structural elements responsible for the allosteric coupling that underlies energy coupling and regulatory
mechanisms. The first system under investigation is KdpFABC, an interesting and unusual hybrid between an
ATP-dependent pump related to P-type ATPases and a K+ channel related to the Superfamily of K+
Transporters. Our previous work has defined the architecture of this heterotetramer and suggests a highly
novel mechanism for transport, in which K+ enters the selectivity filter of the channel-like subunit, travels 40 Å
through a membrane-embedded tunnel, and is then expelled by the pump-like subunit in an energy-dependent
manner. We now plan functional analyses of site-directed mutants to validate this hypothesis and to adopt new
approaches to study the energetics. The second system is YiiP, a Zn2+/H+ antiporter from the Cation Diffusion
Facilitator superfamily. Members of this family form homodimers, have multiple ion binding sites and are
thought to function via an alternating access mechanism. For this system, we have characterized two different
conformations by cryo-EM analysis and MD simulation, and have identified a role for Zn2+ binding on the
transition. We seek to understand better the role of individual Zn2+ binding sites, the nature of occluded states,
the transition between inward- and outward-facing states and the role of protons in this process. We plan also
to study additional members of this family to address the basis for substrate specificity and structural features
that have been postulated to regulate the transport process. In addition to shedding light on these two specific
transport mechanisms, we hope that our work will offer new ways to think about transport that goes beyond
cartoons and structural animations to incorporate protein dynamics and mapping of the energy landscape to
describe the behavior of these molecular machines.

## Key facts

- **NIH application ID:** 10330684
- **Project number:** 1R35GM144109-01
- **Recipient organization:** NEW YORK UNIVERSITY SCHOOL OF MEDICINE
- **Principal Investigator:** David L. Stokes
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $258,657
- **Award type:** 1
- **Project period:** 2022-04-01 → 2027-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10330684

## Citation

> US National Institutes of Health, RePORTER application 10330684, Molecular Mechanisms of Ion Transport (1R35GM144109-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10330684. Licensed CC0.

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