Multistate Structural Modeling and Voltage-Clamp Analysis of Epilepsy/Autism Mutation Kv10.2-R327H Demonstrate the Role of This Residue in Stabilizing the Channel Closed State

Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca2+ concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca2+ regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca2+ concentration, indicating that Ca2+ facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca2+-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca2+ insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca2+, and (b) facilitating closing and stabilization of the closed state in the presence of Ca2+. To explore the role of a negatively charged residue at position 50 in regulation by Ca2+, this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)−. D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES− restores the wild-type Ca2+ regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca2+ regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca2+. Mutant cycle analysis supports a Ca2+-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca2+ destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca2+.

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Voltage clamp analysis of the kinetics of piperidine-induced chloride current in isolated Aplysia neurons

Naunyn-Schmiedeberg s Archives of Pharmacology ◽

10.1007/bf00169048 ◽

1990 ◽

Vol 342 (5) ◽

Cited By ~ 4

Author(s):

K. Takahama ◽

M.R. Klee

Keyword(s):

Voltage Clamp ◽

Chloride Current ◽

Aplysia Neurons ◽

Voltage Clamp Analysis ◽

Kinetics Of

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Voltage clamp analysis of calcium-sensitive chloride current (ICl−Ca) endowed on neuronal membrane in rabbit vesical pelvic ganglia

Neuroscience Research Supplements ◽

10.1016/0921-8696(89)90674-9 ◽

1989 ◽

Vol 9 ◽

pp. 75

Author(s):

Toshihiko Nishimura ◽

Takayuki Tokimasa ◽

Takashi Akasu

Keyword(s):

Voltage Clamp ◽

Chloride Current ◽

Neuronal Membrane ◽

Voltage Clamp Analysis ◽

Pelvic Ganglia

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Voltage Clamp Analysis of Catecholamine-Induced Calcium Conductance Changes in Mammalian Cardiac Muscle

Proceedings in Life Sciences - Biochemistry of Membrane Transport ◽

10.1007/978-3-642-66564-6_34 ◽

1977 ◽

pp. 449-457

Author(s):

H. Reuter

Keyword(s):

Cardiac Muscle ◽

Voltage Clamp ◽

Calcium Conductance ◽

Voltage Clamp Analysis ◽

Conductance Changes

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cAMP-independent effects of 8-(4-parachlorophenylthio)-cyclic AMP on spike duration and membrane currents in pleural sensory neurons of Aplysia

Journal of Neurophysiology ◽

10.1152/jn.1994.72.3.1250 ◽

1994 ◽

Vol 72 (3) ◽

pp. 1250-1259 ◽

Cited By ~ 9

Author(s):

S. Sugita ◽

D. A. Baxter ◽

J. H. Byrne

Keyword(s):

Protein Kinase ◽

Action Potential ◽

Voltage Clamp ◽

Sensory Neurons ◽

Membrane Current ◽

Membrane Currents ◽

Cyclic Monophosphate ◽

Subsequent Application ◽

Independent Effects

1. The serotonergic modulation of pleural sensory neurons in Aplysia is mediated via two second messenger systems: the adenosine cyclic monophosphate/protein kinase A (cAMP/PKA) and diacylglycerol/protein kinase C systems. Often membrane permeable derivatives of cAMP, such as 8-(4-parachlorophenylthio)-cAMP (pcpt-cAMP), have been used to investigate the role of cAMP/PKA in modulating sensory neurons. In light of recent findings that pcpt-cAMP may have cAMP-independent actions, we have reexamined the effects of pcpt-cAMP on the action potential and membrane currents of the sensory neurons. 2. Although pcpt-cAMP (500 microM to 1 mM) and serotonin (5-HT; 10 microM) induced comparable measures of spike broadening (an average increase above baseline of 29 and 40%, respectively), the broadening produced by the two was qualitatively different. Serotonin-induced broadening developed slowly over 9-12 min, was most prominent during later phases of the spike repolarization, and reduced the spike afterhyperpolarization. In contrast, pcpt-cAMP-induced broadening developed rapidly, was rather uniform throughout the repolarization phase of the spike, delayed the peak of the action potential, and increased the afterhyperpolarization. 3. Preexposure of sensory neurons to 5-HT did not occlude further spike broaden by subsequent application of pcpt-cAMP. Indeed the effects of the two were additive. In addition, the effects of pcpt-cAMP were not mimicked by another analogue of cAMP, 8-bromo-cAMP. Interestingly, most of the effects of pcpt-cAMP on the action potential were mimicked by 8-(4-parachlorophenyl-thio)-guanosine cyclic monophosphate (pcpt-cGMP), but not by 8-bromo-cGMP. 4. During voltage-clamp pulses to 20 mV, pcpt-cAMP reduced the membrane current throughout the voltage-clamp pulse, which was qualitatively different from the modulation of the membrane current by 5-HT. In addition, the pcpt-cAMP-induced reduction in the membrane current at the beginning of the pulse was much greater than that induced by 5-HT. Moreover, preexposure of sensory neurons to 5-HT did not occlude further reduction in the membrane current by subsequent application of pcpt-cAMP. 5. These results suggest that pcpt-cAMP has some mechanisms of action that are not shared by 5-HT or cAMP but are shared by pcpt-cGMP. In addition, these findings provide further evidence that results obtained with this compound should be interpreted with caution.

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