Academic Appointments

Honors & Awards

  • Career Development Award (K08), National Institutes of Health (2011)

Professional Education

  • Residency, Stanford Hospital & Clinics, Clinical Pathology (2008)
  • Fellowship, Stanford Hospital & Clinics, Molecular Genetic Pathology (2007)
  • BA, Williams College, Biology & Mathematics (1997)


All Publications

  • Orai1 pore residues control CRAC channel inactivation independently of calmodulin. journal of general physiology Mullins, F. M., Yen, M., Lewis, R. S. 2016; 147 (2): 137-152


    Ca(2+) entry through CRAC channels causes fast Ca(2+)-dependent inactivation (CDI). Previous mutagenesis studies have implicated Orai1 residues W76 and Y80 in CDI through their role in binding calmodulin (CaM), in agreement with the crystal structure of Ca(2+)-CaM bound to an Orai1 N-terminal peptide. However, a subsequent Drosophila melanogaster Orai crystal structure raises concerns about this model, as the side chains of W76 and Y80 are predicted to face the pore lumen and create a steric clash between bound CaM and other Orai1 pore helices. We further tested the functional role of CaM using several dominant-negative CaM mutants, none of which affected CDI. Given this evidence against a role for pretethered CaM, we altered side-chain volume and charge at the Y80 and W76 positions to better understand their roles in CDI. Small side chain volume had different effects at the two positions: it accelerated CDI at position Y80 but reduced the extent of CDI at position W76. Positive charges at Y80 and W76 permitted partial CDI with accelerated kinetics, whereas introducing negative charge at any of five consecutive pore-lining residues (W76, Y80, R83, K87, or R91) completely eliminated CDI. Noise analysis of Orai1 Y80E and Y80K currents indicated that reductions in CDI for these mutations could not be accounted for by changes in unitary current or open probability. The sensitivity of CDI to negative charge introduced into the pore suggested a possible role for anion binding in the pore. However, although Cl(-) modulated the kinetics and extent of CDI, we found no evidence that CDI requires any single diffusible cytosolic anion. Together, our results argue against a CDI mechanism involving CaM binding to W76 and Y80, and instead support a model in which Orai1 residues Y80 and W76 enable conformational changes within the pore, leading to CRAC channel inactivation.

    View details for DOI 10.1085/jgp.201511437

    View details for PubMedID 26809793

  • The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca2+-dependent inactivation. journal of general physiology Mullins, F. M., Lewis, R. S. 2016; 147 (2): 153-164


    The inactivation domain of STIM1 (IDSTIM: amino acids 470-491) has been described as necessary for Ca(2+)-dependent inactivation (CDI) of Ca(2+) release-activated Ca(2+) (CRAC) channels, but its mechanism of action is unknown. Here we identify acidic residues within IDSTIM that control the extent of CDI and examine functional interactions of IDSTIM with Orai1 pore residues W76 and Y80. Alanine scanning revealed three IDSTIM residues (D476/D478/D479) that are critical for generating full CDI. Disabling IDSTIM by a triple alanine substitution for these three residues ("STIM1 3A") or by truncation of the entire domain (STIM11-469) reduced CDI to the same residual level observed for the Orai1 pore mutant W76A (approximately one third of the extent seen with full-length STIM1). Results of noise analysis showed that STIM11-469 and Orai1 W76A mutants do not reduce channel open probability or unitary Ca(2+) conductance, factors that determine local Ca(2+) accumulation, suggesting that they diminish CDI instead by inhibiting the CDI gating mechanism. We tested for functional coupling between IDSTIM and the Orai1 pore by double-mutant cycle analysis. The effects on CDI of mutations disabling IDSTIM or W76 were not additive, demonstrating that IDSTIM and W76 are strongly coupled and act in concert to generate full-strength CDI. Interestingly, disabling IDSTIM and W76 separately gave opposite results in Orai1 Y80A channels: channels with W76 but lacking IDSTIM generated approximately two thirds of the WT extent of CDI but those with IDSTIM but lacking W76 completely failed to inactivate. Together, our results suggest that Y80 alone is sufficient to generate residual CDI, but acts as a barrier to full CDI. Although IDSTIM is not required as a Ca(2+) sensor for CDI, it acts in concert with W76 to progress beyond the residual inactivated state and enable CRAC channels to reach the full extent of inactivation.

    View details for DOI 10.1085/jgp.201511438

    View details for PubMedID 26809794

  • CAP Pathology Resource Guide: Genomics Editors: Cushman-Vokoun, A. M., Abel, G., Arnaout, R., Bry, L., Burton, M. P., Coleman, J. F., Grossman, A., Kaufman, J. H., Kiechle, F. L., Laser, J. S., Mullins, F. M., Santoro, S. A., Thorn, J. College of American Pathologists. Northfield, IL. 2014 (Version 5.0):
  • CAP Pathology Resource Guide: Molecular Pathology Editors: Santoro, S., Arnaout, R., Burton, M. P., Coleman, J. F., Cushman-Vokoun, A. M., Grossman, A., Kiechle, F. L., Laser, J. S., Mullins, F. M. College of American Pathologists. Northfield, IL. 2013 (Version 4.0):
  • CAP Pathology Resource Guide: Genomic Analysis Editors: Santoro, S., Arnaout, R., Bloom, K., Burton, M., Cushman-Vokoun, A., Geiersbach, K., Laser, J., Mullins, F., Robetorye, R. College of American Pathologists. Northfield, IL. 2013 (Version 4.0):
  • Hereditary diffuse gastric cancer due to a previously undescribed CDH1 splice site mutation HUMAN PATHOLOGY Matsukuma, K. E., Mullins, F. M., Dietz, L., Zehnder, J. L., Ford, J. M., Chun, N. M., Schrijver, I. 2010; 41 (8): 1200-1203


    Our patient was a 52-year-old man who was diagnosed with signet ring cell gastric adenocarcinoma. An extensive family history of gastric cancer raised suspicion for hereditary diffuse gastric cancer. Sequencing of the patient's CDH1 gene revealed a novel point mutation in a strictly conserved splice site within intron 6, c.833-2 A > G. This mutation was predicted to result in loss of function due to defective RNA splicing. To characterize the pathogenic mechanism of this mutation, we amplified the patient's CDH1 gene products by reverse transcriptase polymerase chain reaction. Primers flanking the region of the mutation detected 3 distinct transcripts. In addition to the wild-type product, a larger product consistent with activation of a cryptic splice site within intron 6 and a smaller product shown to result from exon 7 skipping were detected. In summary, we have identified a novel CDH1 mutation in a large hereditary diffuse gastric cancer kindred and identified its pathogenic mechanism.

    View details for DOI 10.1016/j.humpath.2010.01.022

    View details for Web of Science ID 000280128300019

    View details for PubMedID 20624523

  • STIM1 and calmodulin interact with Orai1 to induce Ca2+-dependent inactivation of CRAC channels PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mullins, F. M., Park, C. Y., Dolmetsch, R. E., Lewis, R. S. 2009; 106 (36): 15495-15500


    Ca(2+)-dependent inactivation (CDI) is a key regulator and hallmark of the Ca(2+) release-activated Ca(2+) (CRAC) channel, a prototypic store-operated Ca(2+) channel. Although the roles of the endoplasmic reticulum Ca(2+) sensor STIM1 and the channel subunit Orai1 in CRAC channel activation are becoming well understood, the molecular basis of CDI remains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342-448) that binds directly to Orai1 to activate the channel. Surprisingly, CAD-induced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470-491) C-terminal to CAD that is required for CDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orai1, we also investigated a role for CaM in CDI. We identified a membrane-proximal N-terminal domain of Orai1 (residues 68-91) that binds CaM in a Ca(2+)-dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orai1 that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channel inactivation.

    View details for DOI 10.1073/pnas.0906781106

    View details for Web of Science ID 000269632400074

    View details for PubMedID 19706428

  • STIM1 Clusters and Activates CRAC Channels via Direct Binding of a Cytosolic Domain to Orai1 CELL Park, C. Y., Hoover, P. J., Mullins, F. M., Bachhawat, P., Covington, E. D., Raunser, S., Walz, T., Garcia, K. C., Dolmetsch, R. E., Lewis, R. S. 2009; 136 (5): 876-890


    Store-operated Ca(2+) channels activated by the depletion of Ca(2+) from the endoplasmic reticulum (ER) are a major Ca(2+) entry pathway in nonexcitable cells and are essential for T cell activation and adaptive immunity. After store depletion, the ER Ca(2+) sensor STIM1 and the CRAC channel protein Orai1 redistribute to ER-plasma membrane (PM) junctions, but the fundamental issue of how STIM1 activates the CRAC channel at these sites is unresolved. Here, we identify a minimal, highly conserved 107-aa CRAC activation domain (CAD) of STIM1 that binds directly to the N and C termini of Orai1 to open the CRAC channel. Purified CAD forms a tetramer that clusters CRAC channels, but analysis of STIM1 mutants reveals that channel clustering is not sufficient for channel activation. These studies establish a molecular mechanism for store-operated Ca(2+) entry in which the direct binding of STIM1 to Orai1 drives the accumulation and the activation of CRAC channels at ER-PM junctions.

    View details for DOI 10.1016/j.cell.2009.02.014

    View details for Web of Science ID 000263930900011

    View details for PubMedID 19249086

  • Cytologic Evaluation of Lymphadenopathy Associated With Mycosis Fungoides and Sezary Syndrome Role of Immunophenotypic and Molecular Ancillary Studies CANCER CYTOPATHOLOGY Pai, R. K., Mullins, F. M., Kim, Y. H., Kong, C. S. 2008; 114 (5): 323-332


    The most common presenting site of extracutaneous disease in mycosis fungoides and Sezary syndrome is the peripheral lymph node. Although fine-needle aspiration biopsy has been shown to be a valuable diagnostic technique in evaluating lymphadenopathy, its utility in patients with cutaneous T-cell lymphoma has not been extensively studied. With fine-needle aspiration biopsy, material can be collected for ancillary diagnostic studies and for morphologic evaluation.The authors report a series of 11 fine-needle aspiration biopsy specimens from 10 mycosis fungoides and Sezary syndrome patients. Flow cytometric immunophenotyping and T-cell receptor gamma chain polymerase chain reaction were performed on fine-needle aspiration biopsy material and correlated with cytologic findings.Seven of 10 patients had lymph node involvement by cutaneous T-cell lymphoma, with 3 cases exhibiting large-cell transformation and 4 cases exhibiting a small-cell pattern. Flow cytometric immunophenotyping identified an abnormal T-cell population in 6 cases. A clonal T-cell rearrangement by T-cell receptor gamma chain polymerase chain reaction (TCR-gamma PCR) was identified in 1 case in which insufficient events were present for evaluation by flow cytometry and in 1 case in which flow cytometry was not diagnostic of T-cell lymphoma. Two cases showed involvement by classic Hodgkin lymphoma diagnosed by immunohistochemistry on cell block material.Fine-needle aspiration biopsy in conjunction with immunophenotyping and T-cell receptor gamma chain polymerase chain reaction is significantly useful in evaluation of lymphadenopathy in patients with mycosis fungoides and Sezary syndrome, especially for triaging lymph nodes that would otherwise not be sampled or for evaluating multiple lymph nodes.

    View details for DOI 10.1002/cncr.23793

    View details for Web of Science ID 000260140500007

    View details for PubMedID 18798522

  • Identification of an intronic single nucleotide polymorphism leading to allele dropout during validation of a CDH1 sequencing assay: implications for designing polymerase chain reaction-based assays GENETICS IN MEDICINE Mullins, F. M., Dietz, L., Lay, M., Zehnder, J. L., Ford, J., Chun, N., Schrijver, I. 2007; 9 (11): 752-760


    The CDH1 gene encodes the cell adhesion protein E-cadherin, and CDH1 germline mutations are associated with hereditary diffuse gastric cancer. Identification of individuals at high risk of developing diffuse gastric cancer affords the opportunity for endoscopic screening or elective prophylactic gastrectomy. We set out to develop a CDH1 sequencing assay for clinical use.All exons of the CDH1 gene were amplified and sequenced with published and modified primers.While validating the assay, we encountered a case in which a single nucleotide polymorphism located in intron 15 led to allele dropout and therefore to a false-negative result. The polymorphism leading to allele dropout was located within a primer-binding sequence, five bases away from the 3' end of the primer. A frameshift mutation in exon 15 was detected by an alternative primer that binds away from the polymorphic site. A search of the University of California Santa Cruz single nucleotide polymorphism database revealed other polymorphisms located within primer-binding sites. A total of 12 primers in nine primer sets were modified to minimize allele dropout risk.The approach of designing primers to avoid known single nucleotide polymorphisms can be generalized to the design of any polymerase chain reaction-based assay and should be employed whenever possible.

    View details for DOI 10.1097/GIM.0b013e318159a369

    View details for Web of Science ID 000251233500004

    View details for PubMedID 18007144

  • Diagnosis of transfusion-related acute lung injury: TRALI or not TRALI? ANNALS OF CLINICAL AND LABORATORY SCIENCE Fontaine, M. J., MALONE, J., Mullins, F. M., GRUMET, F. C. 2006; 36 (1): 53-58


    TRALI is a challenging diagnosis for both the transfusion specialist and the clinician. A Canadian consensus panel has recently proposed guidelines to better define TRALI and its implications. The guidelines recommend classifying each suspected case in one of the following 3 categories: (1) "TRALI," (2) "Possible TRALI," or (3) "Not TRALI." We report the clinical presentation, laboratory evaluation, and management of 3 patients with respiratory failure (RF) following allogeneic blood transfusions. These patients all experienced RF within 6 hr post-transfusion. Based on a review of the clinical and laboratory data and applying the Canadian guidelines, the first patient, a 67-yr-old man with chronic myelomonocytic leukemia, was diagnosed as "TRALI" due to the sudden onset of RF requiring intensive resuscitation. The second patient, a 55-yr-old man with aplastic anemia, was diagnosed as "Possible TRALI" due to pre-existing RF that worsened after blood transfusion. The third patient, a 1-yr-old male, was diagnosed as transfusion associated circulatory overload (TACO) and "Possible TRALI," although his RF improved after treatment with diuretics. In all 3 cases, the blood donor center was informed of the suspected TRALI reactions. The remaining blood products from the donors associated with these reactions were quarantined. After review of the clinical data, the donors associated with cases #1 and #3 were screened by the blood center for granulocyte and HLA antibodies. Using a Luminex flow bead array, the following class I and class II antibodies specific for patient #1 were identified in the respective donor: anti-A25, B8, B18, and anti-DR15, DR 17. Subsequently, donor #1 was permanently deferred. A non-specific IgM anti-granulocyte antibody was identified in the donor associated with case #3, and this donor was subsequently disqualified from plasma and platelet donations. In conclusion, the Canadian guidelines to categorize patients suspected of TRALI provide a useful framework for evaluation of these patients and their respective blood donors.

    View details for Web of Science ID 000236733400008

    View details for PubMedID 16501237

  • Functional interaction between extracellular sodium, potassium and inactivation gating in HERG channels JOURNAL OF PHYSIOLOGY-LONDON Mullins, F. M., Stepanovic, S. Z., Gillani, N. B., George, A. L., Balser, J. R. 2004; 558 (3): 729-744


    We have studied the interaction between extracellular K(+) (K(+)(o)) and extracellular Na(+) (Na(+)(o)) in human ether-à-go-go related gene (HERG)-encoded K(+) channels expressed in Chinese hamster ovary (CHO-K1) cells, using the whole-cell voltage clamp technique. Prior studies indicate that Na(+)(o) potently inhibits HERG current (IC(50) 3 mm) by binding to an outer pore site, and also speeds recovery from inactivation. In this study, we sought to explore the relationship between the Na(+)(o) effect on recovery and Na(+)(o) inhibition of HERG current, and to determine whether inactivation gating plays a critical role in Na(+)(o) inhibition of HERG current. Na(+)(o) concentration-response relationships for current inhibition and speeding of recovery were different, with Na(+)(o) less potent at speeding recovery. Na(+)(o) inhibition of HERG current was relieved by physiological [K(+)](o), while Na(+)(o) speeded recovery from inactivation similarly in the absence or presence of physiological [K(+)](o). To examine the link between Na(+)(o) block and inactivation using an independent approach, we studied hyperpolarization-activated currents uncoupled from inactivation in the S4-S5 linker mutant D540K. Depolarization-activated D540K currents were inhibited by Na(+)(o), while hyperpolarization-activated currents were augmented by Na(+)(o). This result reveals a direct link between Na(+)(o) inhibition and a depolarization-induced conformational change, most likely inactivation. We attempted to simulate the disparate concentration-response relationships for the two effects of Na(+)(o) using a kinetic model that included Na(+)(o) site(s) affected by permeation and gating. While a model with only a single dynamic Na(+)(o) site was inadequate, a model with two distinct Na(+)(o) sites was sufficient to reproduce the data.

    View details for DOI 10.1113/jphysiol.2004.065193

    View details for Web of Science ID 000223435900002

    View details for PubMedID 15169846

  • Extracellular sodium interacts with the HERG channel at an outer pore site JOURNAL OF GENERAL PHYSIOLOGY Mullins, F. M., Stepanovic, S. Z., Desai, R. R., George, A. L., Balser, J. R. 2002; 120 (4): 517-537


    Most voltage-gated K(+) currents are relatively insensitive to extracellular Na(+) (Na(+)(o)), but Na(+)(o) potently inhibits outward human ether-a-go-go-related gene (HERG)-encoded K(+) channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429-30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na(+) (Na(+)(i)) and extracellular Ba(2+) (Ba(2+)(o)), to define a site where Na(+)(o) interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na(+)(o) was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K(+) selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na(+)(o). Quantitatively similar effects of Na(+)(i) to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na(+)(o) sensitivity, this result suggested that Na(+)(o) and Na(+)(i) act at different sites. Extracellular Ba(2+) (Ba(2+)(o)) blocks K(+) channel pores, and thereby serves as a useful probe of K(+) channel structure. HERG channel inactivation promotes relief of Ba(2+) block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265-278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na(+)(o) action. A remote allosteric model predicts that Na(+)(o) will speed relief of Ba(2+)(o) block by promoting inactivation. Instead, Na(+)(o) slowed Ba(2+) egress and Ba(2+) relieved Na(+)(o) inhibition, consistent with Na(+)(o) binding to an outer pore site. The apparent affinities of the outer pore for Na(+)(o) and K(+)(o) as measured by slowing of Ba(2+) egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na(+)(o) inhibition. Na(+)(o) inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A.

    View details for DOI 10.1085/jgp.20028589

    View details for Web of Science ID 000178529200009

    View details for PubMedID 12356854

  • Probing the interaction between inactivation gating and D-sotalol block of HERG CIRCULATION RESEARCH Numaguchi, H., Mullins, F. M., Johnson, J. P., Johns, D. C., Po, S. S., Yang, I. C., Tomaselli, G. F., Balser, J. R. 2000; 87 (11): 1012-1018


    Potassium channels encoded by HERG underlie I:(Kr), a sensitive target for most class III antiarrhythmic drugs, including methanesulfonanilides such as Dd-sotalol. Recently it was shown that these drugs are trapped in the channel as it closes during hyperpolarization. At the same time, HERG channels rapidly open and inactivate when depolarized, and methanesulfonanilide block is known to develop in a use-dependent manner, suggesting a potential role for inactivation in drug binding. However, the role of HERG inactivation in class III drug action is uncertain: pore mutations that remove inactivation reduce block, yet many of these mutations also modify the channel permeation properties and could alter drug affinity through gating-independent mechanisms. In the present study, we identify a definitive role for inactivation gating in Dd-sotalol block of HERG, using interventions complementary to mutagenesis. These interventions (addition of extracellular Cd(2+), removal of extracellular Na(+)) modify the voltage dependence of inactivation but not activation. In normal extracellular solutions, block of HERG current by 300 micromol/L Dd-sotalol reached 80% after a 10-minute period of repetitive depolarization to +20 mV. Maneuvers that impeded steady-state inactivation also reduced Dd-sotalol block of HERG: 100 micromol/L Cd(2+) reduced steady-state block to 55% at +20 mV (P:<0.05); removing extracellular Na(+) reduced block to 44% (P:<0.05). An inactivation-disabling mutation (G628C-S631C) reduced Dd-sotalol block to only 11% (P:<0.05 versus wild type). However, increasing the rate of channel inactivation by depolarizing to +60 mV reduced Dd-sotalol block to 49% (P:<0.05 versus +20 mV), suggesting that the drug does not primarily bind to the inactivated state. Coexpression of MiRP1 with HERG had no effect on inactivation gating and did not modify Dd-sotalol block. We postulate that Dd-sotalol accesses its receptor in the open pore, and the drug-receptor interaction is then stabilized by inactivation. Whereas deactivation traps the bound methanesulfonanilide during hyperpolarization, we propose that HERG inactivation stabilizes the drug-receptor interaction during membrane depolarization.

    View details for Web of Science ID 000165607100012

    View details for PubMedID 11090546

  • Human ether-a-go-go-related gene K+ channel gating probed with extracellular Ca2+ - Evidence for two distinct voltage sensors JOURNAL OF GENERAL PHYSIOLOGY Johnson, J. P., Mullins, F. M., Bennett, P. B. 1999; 113 (4): 565-580


    Human ether-à-go-go-related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 +/- 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go-related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery.

    View details for Web of Science ID 000079648200005

    View details for PubMedID 10102937