Electrophysiology of Membrane Potentials

Lecture 011625

• Bioelectric circuits and ion channels are studied using electrophysiological approaches, including molecular studies, and explore channel types and their gating mechanisms.

• Membrane potential is influenced by electrochemical equilibrium, resting membrane potential, selective membrane permeability, and the Nernst and Goldman equations. It's also influenced by electrotonic (passive) properties, including time and length constants.

• The action potential is a key concept introduced in the lecture.

Membrane Potentials

• Changes in membrane potential (VM) from rest include graded responses (synaptic potentials) and active responses (action potentials).

• Resting membrane potential is negative inside relative to outside the cell.

Study of Ion Channels - Electrophysiology

• Intracellular recording measures membrane potential (VM) via current clamp.
• Patch clamp recording measures ionic current (IK+ , INa+ , ICa++).
• Conventions for recordings: depolarization (upward deflection) and hyperpolarization (downward deflection) for voltage; inward current (downward deflection) and outward current (upward deflection) for current.
• Membrane potential = VM, Ionic current = I(ION).

Intracellular recording of resting and action potentials

• Injecting positive current depolarizes the membrane, while injecting negative current hyperpolarizes it.
• Reaching a threshold level of depolarization triggers action potentials.

Patch clamp recording of single voltage-gated Na+ channel

• Membrane depolarization opens voltage-gated sodium channels, allowing Na+ influx.
• This process is crucial to ionic current flow as part of the action potential.

Nerve cell membrane

• Lipid bilayer forms a barrier to ion diffusion.
• Pumps actively transport ions to create concentration gradients.
• Ion channels are embedded in the phospholipid bilayer and selectively permit the passage of specific ions.
• Channels are gated by various stimuli.

Types of Ion Channels

• Biochemical-gated channels are regulated by G proteins, cyclic nucleotides, or intracellular Ca2+.
• Ligand-gated channels open in response to neurotransmitters binding.
• Voltage-gated channels open in response to changes in membrane potential.
• Mechanosensitive channels open in response to mechanical stimuli.

Intracellular recording of resting membrane potential

• The resting membrane potential is the difference in electrical potential across a cell membrane during its resting phase, typically ranging from -40mV to -100mV.
• The oscilloscope reading represents the charge difference.

Basis of Membrane Potential

• The sodium-potassium pump establishes ion concentration gradients (high Na+ outside, high K+ inside).
• Its electrogenic nature contributes slightly to the resting membrane potential (about -5mV to -10mV).
• Ion flux through open ion channels is the primary contributor to the resting membrane potential (-75mV).

Role of Ion Channels

• Electrochemical equilibrium involves concentration and electrical gradients.
• The equilibrium potential for an ion (Eion) depends on the ion's concentration gradient.
• Selective permeability (PION) is crucial in determining membrane potential using the Goldman-Hodgkin-Katz equation (EM = Σ[PION x EION]).

Resting Membrane Potential vs Equilibrium Potential

• Resting membrane potential (EM) is the sum of all equilibrium potentials (EION) considering ion permeability (PION).
• Key ions contributing to resting potential are primarily K+ and, to a lesser degree, Na+ and Cl-.

Electrotonic (passive) membrane properties

• Time constant (τ) measures how quickly a membrane potential changes in response to a stimulus.
• Length constant (λ) measures how far a change in membrane potential spreads along the membrane.

Study of Ion Channels - X-ray Crystallography

• Structures of ion channels like KcsA (bacterial K+ channel) are determined via X-ray crystallography or cryo-EM for eukaryotic channels.

Study of Ion Channels - Molecular Biology

• Gene sequences reveal the association of subunits with cell membranes to form channels and pores within cells.
• Hydrophobicity-based plots from the gene sequences help in understanding channel subunit structures, including location of hydrophobic and hydrophilic domains.

Using patch clamp to study ion channels

• Xenopus oocyte expression system is used for studying ion channel function and to compare normal channels to mutated ion channels that cause disease.

Na and K channel responses to VM

• Na+ channels open and then inactivate in response to depolarization, while K+ channels open in response to depolarization to drive repolarization.

Expression of Na+ channels in frog egg

• The frog oocyte expression system to insert mRNA for channel proteins enables the study of particular ion channels in isolation.

Channelopathies

• Various genetic disorders (channelopathies) can be caused by mutations in ion channel genes in muscle and brain tissue, which affect their function, such as in hyperkalemic periodic paralysis, paramyotonia congenita, hypokalemic periodic paralysis, generalized epilepsy with febrile seizures, severe myoclonic epilepsy of infancy, or idiopathic childhood epilepsy.

Ion Channels as Conductors of Electricity

• Ohmic channels follow Ohm's law (V = iR).
• Rectifying (non-ohmic) channels deviate from Ohm's law, with conductance varying depending on the direction of current.

Action Potentials

• Action potentials are rapid changes in membrane potential that travel along axons and trigger muscle contraction.
• Frequency coding represents the intensity of a stimulus.
• Recruitment of axons carries information about the stimulus.
• Place code indicates which axons contribute to the stimulus.
• Voltage-gated Na+ and K+ channels are crucial for the rapid, faithful propagation of action potentials.