Research

We aim to develop mechanistic insights into how nerve terminals precisely control Ca²⁺-evoked neurotransmitters release over wide timescales and modify the strength and efficacy of this process in an actively dependent manner. Recent structural and functional analyses have provided important insights into the molecular organization of pre-synaptic vesicular release machinery and have prompted several molecular models (Rothman, Krishnamkumar et al. FEBS Letters, 2017; Volynski and Krishnakumar, Curr Opin Neurobiol, 2018) to explain the mechanics of synaptic vesicle priming, calcium-evoked release, and short-term plasticity. Using these models as starting points, we are working to delineate the molecular mechanism of fast synchronous and delayed asynchronous release and understanding how distinct calcium sensors synergistically regulate different models of evoked exocytosis. We are also working to understand how pre-synaptic Ca²⁺ dynamics tune vesicle docking, priming and fusion process to facilitate plasticity of neurotransmitter disease.

To this end, we employ multidisciplinary biochemical, biophysical, and high-resolution structural methods, with a specific focus on systematic reconstitution strategies. Combining a recently developed in vitro single-vesicle fusion setup (Ramakrishnan et al. Elife, 2020; Bera et al. Elife, 2022) with calcium uncaging paradigms, we are working to reconstitute different models of calcium-evoked release and short-term plasticity with millisecond precision under cell-free conditions. Our approach is to use in vitro functional analysis with isolated proteins to guide physiological analysis in cultured neurons (in collaboration with professor Kirill Volynski, PhD, University College London, Queens Square Institute of Neurology).

This is a collaborative research program that is aimed at uncovering the molecular and mechanistic basis for pre-synaptic synaptopathies. We use reconstituted fusion systems, combined with biochemical tools to examine the functional effect of both de novo mutations and neuropathy-associated genetic variants in core pre-synaptic proteins (Coleman et al. Cell Reports, 2018; Salpietro et al. American Journal Human Genetics, 2019). This approach enables us to rigorously and rapidly screen the consequence of mutations/alterations in individual synaptic proteins (or any epistatic effects) on neurotransmitter release and use these "experiments-of-nature" mutations to gain insight into the mechanism of synaptic transmission. We aim to drive rapid translation of the in vitro work towards neuronal cultures/animal models (in collaboration) to establish physiological correlation.

We are currently focusing on paroxysmal neurological disorders like epilepsy and migraine, and have established successful collaborations with Experimental Epilepsy groups, particularly those of Kirill Volynski and Henry Houlden, PhD, both of University College London, Queens Square Institute of Neurology. We are exploring new, collaborative opportunities both within and outside of Yale in order to extend the analysis to other associated neurological, neurodevelopmental, and psychiatric disorders.