A channelopathy mutation in the voltage-sensor discloses contributions of a conserved phenylalanine to gating properties of Kv1.1 channels and ataxia

Scientific Reports, Jul 2017

Channelopathy mutations prove informative on disease causing mechanisms and channel gating dynamics. We have identified a novel heterozygous mutation in the KCNA1 gene of a young proband displaying typical signs and symptoms of Episodic Ataxia type 1 (EA1). This mutation is in the S4 helix of the voltage-sensing domain and results in the substitution of the highly conserved phenylalanine 303 by valine (p.F303V). The contributions of F303 towards K+ channel voltage gating are unclear and here have been assessed biophysically and by performing structural analysis using rat Kv1.2 coordinates. We observed significant positive shifts of voltage-dependence, changes in the activation, deactivation and slow inactivation kinetics, reduced window currents, and decreased current amplitudes of both Kv1.1 and Kv1.1/1.2 channels. Structural analysis revealed altered interactions between F303V and L339 and I335 of the S5 helix of a neighboring subunit. The substitution of an aromatic phenylalanine with an aliphatic valine within the voltage-sensor destabilizes the open state of the channel. Thus, F303 fine-tunes the Kv1.1 gating properties and contributes to the interactions between the S4 segment and neighboring alpha helices. The resulting channel’s loss of function validates the clinical relevance of the mutation for EA1 pathogenesis.

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A channelopathy mutation in the voltage-sensor discloses contributions of a conserved phenylalanine to gating properties of Kv1.1 channels and ataxia

Scientific RepoRts | A channelopathy mutation in the voltage-sensor discloses contributions of a conserved phenylalanine to gating properties of Kv1.1 channels and ataxia Sonia Hasan 2 Cecilia Bove 1 Gabriella Silvestri 0 Elide Mantuano 4 Anna Modoni 0 Liana Veneziano 4 Lara Macchioni 1 Therese Hunter 3 Gary Hunter 3 Mauro Pessia 1 3 Maria Cristina D'Adamo 3 0 Institute of Neurology, Catholic University of Sacred Heart, Fondazione Gemelli , Rome , Italy 1 Section of Physiology and Biochemistry, Department of Experimental Medicine, School of Medicine, University of Perugia , Perugia , Italy 2 Department of Physiology, Faculty of Medicine, Kuwait University , Safat, 13110 , Kuwait 3 Faculty of Medicine & Surgery, Department of Physiology & Biochemistry, University of Malta , MSD 2080, Msida , Malta. Sonia Hasan and Cecilia 4 Institute of Translational Pharmacology, National Research Council of Italy , Rome , Italy OPEN Published: xx xx xxxx Channelopathy mutations prove informative on disease causing mechanisms and channel gating dynamics. We have identified a novel heterozygous mutation in the KCNA1 gene of a young proband displaying typical signs and symptoms of Episodic Ataxia type 1 (EA1). This mutation is in the S4 helix of the voltage-sensing domain and results in the substitution of the highly conserved phenylalanine 303 by valine (p.F303V). The contributions of F303 towards K+ channel voltage gating are unclear and here have been assessed biophysically and by performing structural analysis using rat Kv1.2 coordinates. We observed significant positive shifts of voltage-dependence, changes in the activation, deactivation and slow inactivation kinetics, reduced window currents, and decreased current amplitudes of both Kv1.1 and Kv1.1/1.2 channels. Structural analysis revealed altered interactions between F303V and L339 and I335 of the S5 helix of a neighboring subunit. The substitution of an aromatic phenylalanine with an aliphatic valine within the voltage-sensor destabilizes the open state of the channel. Thus, F303 fine-tunes the Kv1.1 gating properties and contributes to the interactions between the S4 segment and neighboring alpha helices. The resulting channel's loss of function validates the clinical relevance of the mutation for EA1 pathogenesis. - Voltage-gated potassium channels (Kv) play key roles in neurotransmission and nerve cell physiology1 and are of high therapeutic relevance2. Strategically clustered at critical subdomains such as the axon initial segment3, juxtaparanodal regions of the Ranvier’s node4, as well as in synaptic nerve terminals5, Kv1 channels are known key regulators of excitability. They open at voltages close to action potential (AP) threshold to reduce excitability by limiting nodal re-excitation6, shaping the presynaptic AP waveform and controlling transmitter release at synaptic terminals7. Accordingly, mutations that result in dysfunctional Kv1.1 channels are likely to result in impairments in neuronal excitability and neurotransmission. Of particular clinical importance is the Kv1.1 channelopathy that is predominantly linked to Episodic Ataxia type 1 (EA1). EA1 is an autosomal dominant disorder characterized by frequent, albeit brief, attacks of uncoordinated movements (ataxia) and involuntary repetitive muscular contractions (myokymia). The disorder presents primarily by means of heterozygous point mutations in the KCNA1 (Kv1.1) gene, located on chromosome 12p138–10. Kv1.1 co-assembles with α-subunits of other members of the Kv1 family to form heterotetrameric channels with biophysical and pharmacological properties that are distinct from homotetramers made up of their contributing subunits11–16. Both in the periphery17, 18 and in the brain19, 20 Kv1.1 is mostly found co-assembled with Kv1.2 subunits. A mutation in Kv1.1 affects the function of the Kv1.1/1.2 heteromeric channel to which they contribute21. A number of functional studies on Kv1.1 mutations all report loss of function of the channel1, 8, 21–29. Yet, despite this consensus, phenotypic heterogeneity such as severity of ataxia and presence of additional features such as epilepsy and hyperthermia exist24, 30, 31 and may be connected to differences in mechanisms of dysfunction, altered specific channel parameters or epigenetics30, 32–34. Structurally, Kv channels are composed of four subunits each of which contains six transmembrane-spanning segments (S1 through S6) with cytoplasmic N- and C-terminal domains (Fig. 1c). The S1–S4 segments comprise the voltage-sensing domain, which senses the membrane potential and controls the gating of the pore domain (S5–S6). Kv1.1 α-subunits make up channels that mediate the non-inactivating outward delayed-rectifier potassium current14. The positively charged amino acids in the S4 segment play a central role in the voltage-dependence of Kv1.1 channels. In particular, the characteristic low activation threshold and distinct activation-deactivation kinetics features (...truncated)


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Sonia Hasan, Cecilia Bove, Gabriella Silvestri, Elide Mantuano, Anna Modoni, Liana Veneziano, Lara Macchioni, Therese Hunter, Gary Hunter, Mauro Pessia, Maria Cristina D’Adamo. A channelopathy mutation in the voltage-sensor discloses contributions of a conserved phenylalanine to gating properties of Kv1.1 channels and ataxia, Scientific Reports, 2017, Issue: 7, DOI: 10.1038/s41598-017-03041-z